Born February 15, 2484 on Vesselside (possibly a euphemism meaning "aboard a spaceship"), Zoë served in the Unification War under Sergeant Malcolm Reynolds and continues under his command on his spaceship, Serenity. She is a loyal second-in-command and a tough, deadly fighter. She married Serenity's pilot, Wash, sometime after he joined the crew, though initially she claimed that something about him "bugged" her — possibly his disturbing moustache (seen in flashback in the episode "Out of Gas"). In the DVD commentary for the episode "Shindig", costume designer Shawna Trpcic mentions that the leather necklace Zoë always wears is a symbol of her marriage bond. However, she is seen wearing the necklace in the flashback sequences of "Out of Gas", well before she has married Wash.

According to the Joss Whedon book Serenity: The Official Visual Companion, Zoë's last name, at least at the time of the Unification War, was Alleyne and she took the name Washburne after her marriage to Hoban Washburne. This is confirmed in a deleted scene from Serenity where a display lists her military name as Corporal Zoë Alleyne.

At the time of the Battle of Serenity Valley, Zoë had attained the rank of Corporal. Along with Sergeant Malcolm Reynolds, she was the only other survivor of that particular battle for their assigned unit, the 57th Overlanders.

Unlike Mal, she was career military, and is often more calm in dangerous situations than Mal (possibly a difference between being volunteer and career military). Mal often thinks with his heart whilst Zoë often thinks with her head. Regardless, Zoë follows all orders from Mal, although often with a degree of advice or concern.

Her weapon of choice appears to be identical to the Mare's Leg, a custom Winchester Model 1892 rifle used by Steve McQueen's character, Josh Randall, in the television series Wanted: Dead or Alive. Joss Whedon, creator of Firefly, calls the Mare's Leg his "favourite gun of all times".

She is the only member of Mal's crew to call him "sir" and take orders as if receiving them from a superior officer.