Two women, aged 33 and 30-years-old, were working at the Ladbrokes branch on East Main Street, Darvel, on Thursday when the man entered.
He spoke with a Northern Irish accent and is described as white, 6ft, early 30s, slim build with dark brown hair and facial hair. He was wearing a blue hooded top with the hood up, black trousers, black and white training shoes and gloves at the time.
He was last seen walking in the around the Cross Street and West Donnington Street area.
Police confirmed that while shop staff were distressed by the incident, no one was injured.
Detective Inspector Michael Carr, at Kilmarnock CID, told the Evening Times: “This was a very distressing experience for the two members of staff who fortunately were not physically injured.
“The area would have been busy with local residents and commuters at the time of the incident and I’d like to speak to anyone who may have seen the culprits hanging around prior to the robbery taking place or leaving the premises afterwards.
“I also urge anyone who has dash cam footage that may have captured the suspects in the area to contact police immediately.”
Anyone with information can contact Kilmarnock CID via 101 or Crimestoppers on 0800 555 111 where anonymity can be maintained.