Ayame Sarutobi, frequently called Sa-chan or Sacchan, is a kunoichi and a former elite member of the Oniwabanshuu, the shogunate's secret police force. She is a young woman with long, flowing purple hair, grey eyes, and a distinctive beauty mark, or tear mole, beneath her right eye. Her most defining physical feature is her red-framed glasses, which are a necessity due to her exceptionally poor eyesight. Without them, she is functionally blind, unable to distinguish people from inanimate objects, and has mentioned that her hearing also becomes less reliable. Despite this severe myopia, she is a highly capable ninja.

Her personality is marked by a sharp and often contradictory duality. On the one hand, she is a consummate professional, trained from childhood by the leader of the Oniwabanshuu, Hattori Zenzou's father. In this capacity, she operates as a freelance assassin or a shimatsuya, a cleaner who disposes of criminals and individuals beyond the reach of the law. She is fiercely loyal, possesses a strong sense of justice, and takes great pride in her work, executing missions with lethal precision. On the other hand, in her personal life, she is loud, boisterous, and overwhelmingly fixated on the samurai Sakata Gintoki. This fixation manifests as extreme masochism; she derives pleasure from being verbally abused and physically mistreated by him, interpreting his harsh words and violent reactions as signs of affection. She is an unabashed stalker, frequently lurking around the Yorozuya office and hiding in improbable places to watch him. Her love for Gintoki is obsessive but genuine, having blossomed after he saved her life despite her initial attempt to deceive him.

Ayame's motivations are primarily driven by her feelings for Gintoki and her sense of duty. Her desire to be near him dictates much of her free time, leading to her constant stalking and her comedic attempts at seduction, which often involve suggestive poses or tying herself up. However, when her work as an assassin or her loyalty to the shogunate is at stake, she switches to a serious, focused killer mode, prioritizing the mission above all else, even her obsession. She has a noted rivalry with Shimura Tae and Tsukuyo, whom she views as romantic rivals for Gintoki's attention, leading to frequent squabbles. Her relationship with her childhood friend and fellow Oniwabanshuu trainee, Hattori Zenzou, is complex; she constantly bullies him, often targeting his hemorrhoids with her kunai, yet she deeply respects him and risked her life to save him during the Shogun Assassination arc. She shares a loyal bond with the former Shogun, Tokugawa Shigeshige, having sworn to protect him since childhood.

Within the story, Ayame serves as both a source of recurring comedy and a formidable ally during serious arcs. Her comedic role is that of an overly aggressive masochist and stalker, whose appearances often involve her being violently rejected by Gintoki or engaging in absurd schemes. Her role becomes more significant in dramatic storylines, particularly the Shogun Assassination arc, where she reunites with her former Oniwabanshuu comrades to protect the Shogun. She demonstrates her true abilities, exposing hidden enemies and fighting fiercely aboard a hostile ship. While her primary development is more a deepening of her established traits than a fundamental change, her loyalty and capacity for serious action are consistently shown. In moments of crisis, she sheds her comedic persona to reveal a dependable and brave warrior. Her love for Gintoki remains a constant, but it is portrayed with increasing sincerity, showing that beneath her perverse exterior lies a deeply devoted and caring individual.

As a ninja, Ayame's notable abilities are considerable. She is exceptionally agile, capable of running across rooftops and leaping between buildings with ease. Her skill with thrown kunai is almost perfect; ironically, her aim remains lethal even when she is not wearing her glasses. She is proficient in various ninja tools, including traps, smoke bombs, and caltrops, and is known to use her favorite food, sticky fermented soybeans called natto, as an unconventional weapon. She is a master of stealth and infiltration, skills she unfortunately uses more often to stalk Gintoki than for her professional duties. Despite her many talents, her poor eyesight and her emotional instability when it comes to Gintoki remain her most persistent weaknesses.