Extra training of pub staff, as part of a mandatory code of practice, could cost the drinks trade between £37.7m and £151m in its first year, according to government estimates.
Pubs could be made to offer staff extra government-accredited training to make them aware of all laws around selling alcohol, which is part of the code, unveiled in last month's Queen's Speech.
The cost of training in subsequent years, with a 50 per cent turnover of staff, would cost between £19m and £76m, the government predicts.
Commenting on the costs, Jeremy Allen, of licensing solicitors Poppleston Allen, said: "Surely it's hardly the right time to impose such an additional burden on the licensed trade."
He added that overall the most concerning aspect of the new rules is the power the Home Secretary will have to specify certain conditions, which can then be added to all relevant premises.
"Most worryingly of all, this does not just relate to new licences," he added. "In other words, you have acquired premises which are currently trading and the Secretary of State's conditions suddenly apply. What's more, if you have an existing condition, but the Secretary of State's is more onerous, the new one applies.
"What's more, there doesn't appear to be any right of appeal against any of the conditions imposed. It may be possible to challenge local Authorities, but we shall have to wait and see.
"It is frightening if your Local Authority imposes strict conditions whereas the neighbouring one, just down the road from you, doesn't."