The Thomson TUI shop in St Sepulchre Gate has taken the security measures after a recent spate of armed raids on travel agents in the region.
Customers visiting the shop now have to alert staff inside before being let in.
A TUI UK spokesman said: “We take the safety of our customers and colleagues extremely seriously at all times and always ensure we have appropriate measures in place across our operations.
"We have taken the decision to put additional safety processes in place, such as having a locked door policy, in some of our stores in the Yorkshire area due to recent reported incidents.”
In recent months, there have been a number of armed raids on travel agents in Sheffield, with four in the last few year - although the incidents are not thought to be linked.
In the most recent attack staff were left terrified after two people, both with their faces covered, burst into a branch of Thomas Cook in Hillsborough, Sheffield with one of them brandishing a machete.
They threatened staff before fleeing the scene with a quantity of cash from the till in a white coloured getaway car.
In July last year, two men armed with guns raided a travel agents in Chapeltown and a month later in August, two men - reportedly armed with a sawn-off shotgun and a sledgehammer - threatened staff at the Thomson outlet, now called TUI, in Hillsborough, and fled with cash.
And in March this year staff at Hays travel agents in Chapeltown were left 'traumatised and shaken' after a gang burst in with an axe, machete and a knife.