The robbery took place in the Ladbrokes bookies in the Maybole area on Sunday at around 5.35pm with detectives appealing for information.
A member of staff was threatened with a weapon as the suspect demanded money. After being given the money he drove off in a light coloured saloon car heading towards John Knox Street.
The suspect, who was wearing a dark jacket with the hood up and light coloured trousers, is described as being in his early 40s with stubble.
Fraser Normansell, detective inspector Ayr CID, said: “I would appeal to anyone who was in the surrounding area yesterday evening and may have seen anything suspicious to please get in touch.
“Anyone with information is asked to contact Kilmarnock CID via 101 and quote incident number 3261 of Sunday 8th October 2017. Alternatively you can call CRIMESTOPPERS on 0800 555 111 where details can be given in confidence.”