The 39-year-old signed his contract on Thursday to join former Hearts teammate Allan Johnston’s management team.
Locke, who has been without a job since he was replaced by Robbie Neilson as Hearts manager last month, played for Killie for seven years before becoming a coach under Jim Jefferies and Billy Brown.
Kilmarnock chairman Michael Johnston told STV: “I’m delighted to welcome Gary back to Rugby Park. He was a very popular figure and a great leader in the dressing room during his seven years as a player with Kilmarnock and those qualities, together with his recent management experience in the Premiership, made him our principal target for the post of assistant manager.”
He added that he felt Locke and his existing manager would make a great partnership due to their many years as players together. He said:
“Allan Johnston and Gary have spent many years together as youth and senior players in the past and share a mutual understanding of what’s required to bring success to Rugby Park. They are both ‘winners’ by nature - every match will be a contest they want to win.”
A former Hearts captain, he was not offered a new contract as Hearts manager when new owner Ann Budge took control at Tynecastle last month.
Kilamrnock had been without an assistant boss since Sandy Clark’s departure last month.