Ayame Sarutobi, most often called Sacchan or Sa-chan by those who know her, is a kunoichi, or female ninja, who originally trained as a member of the shogunate's elite Oniwabanshuu unit. Following the disbandment of this organization, she began working as a freelance assassin who targets corrupt individuals, and she also holds a part-time job at the Kunoichi Café. She has long, light purple hair and a beauty mark situated under her right eye. Her typical attire consists of a modified martial arts training top with cut-off sleeves, a red waistband, dark purple forearm sleeves, and a matching scarf. She stands 169 centimeters tall and has a noted physical figure that is occasionally remarked upon by other characters.

The most defining and immediately noticeable trait of Sacchan is her extremely poor eyesight. She is severely myopic to the point of being nearly blind without her distinctive dark-red glasses, unable to reliably differentiate between living beings and inanimate objects. This visual impairment is a constant source of comedic mishaps, often leading her to mistake other people, such as Kagura disguised as Gintoki, for her object of affection. Her glasses, which she inherited from her late grandmother, hold significant emotional value for her beyond their practical necessity.

In terms of personality, Sacchan is a character of dramatic and often contradictory extremes. On one hand, she can be loud, boisterous, and openly perverted, frequently screaming or screeching when excited. On the other hand, when engaged in her professional duties, she becomes grave, focused, and highly competent, showing a level of seriousness that contrasts sharply with her usual demeanor. Her most pronounced and persistent personality trait, however, is an obsessive, masochistic infatuation with the samurai Sakata Gintoki. This obsession began after her first encounter with him, where he saved her despite her having lied to him. Since that moment, she has devoted herself to stalking him, often hiding in improbable locations near his office and interpreting his aggressive insults and physical violence as forms of affectionate attention. This dynamic has awakened a masochistic side to her personality that is exclusively directed at Gintoki, and she relishes the harsh treatment he gives her.

Despite her seemingly frivolous nature, Sacchan is motivated by a strong sense of loyalty and duty. She holds a deep, solemn commitment to protecting the former shogun, Tokugawa Shigeshige, having known him since childhood and sworn to guard him. This loyalty also extends to her fellow former Oniwabanshuu members, particularly Hattori Zenzou. Although their relationship is characterized by her constantly bullying him and targeting his hemorrhoids in comedic fashion, she recognizes him as a capable leader and is willing to risk her life to save him in serious situations, demonstrating a profound underlying bond. Her relationships with other female characters are similarly complex. She views Shimura Tae as a primary romantic rival, leading to frequent comedic clashes, yet she has shown genuine care for her, such as when she tearfully urged a hospitalized Tae to fight an illness. She also initially bickers with the courtesan Tsukuyo, another rival for Gintoki's affection, but the two eventually develop a friendship, with Sacchan affectionately calling her Tsukki.

Sacchan's role in the story is primarily that of a recurring comedic foil for Gintoki and the Yorozuya, but she also participates in major serious arcs. In the Shogun Assassination Arc, she reunites with the remnants of the Oniwabanshuu to protect the shogun, fighting fiercely aboard an enemy vessel and sustaining injuries. Her development, while subtle, shows that her fixation on Gintoki is not entirely one-dimensional. In the film Be Forever Yorozuya, she refuses to cut her hair and maintains her appearance for five years, firmly believing that Gintoki is still alive and will return, highlighting a more sincere and patient devotion beneath her obsessive exterior. She has also demonstrated growth in understanding the value of friendship over material objects, as seen when she comes to terms with the loss of a pair of glasses given to her by Gintoki.

As a combatant, Sacchan possesses extraordinary physical skills and is a master of ninjutsu. Her most notable ability is her perfect aim with kunai, which she can throw with pinpoint accuracy even when not wearing her glasses. Her fighting style is unconventional and includes the use of natto, a sticky fermented bean dish, which she employs to trap and immobilize her enemies. She also demonstrates high agility, enhanced reflexes, and a remarkable tolerance for pain. On one occasion, her glasses were temporarily upgraded by the inventor Gengai to include features such as laser beams and a protective angel mode, allowing her to perform a technique known as the Flight of Hundred Glasses. Despite her poor eyesight being a persistent weakness, her overall combat prowess makes her a formidable assassin and a valuable ally in battle.