The fees were increased to 36.40 liras ($ 6.08) and 54.70 liras ($ 9.14) for cars and minibuses respectively, from 23.30 ($ 3.90) and 34.90 liras ($ 5.83).
Last year, a similar price hike to the tunnel fees was backtracked and the ministry had announced that the new chart had been put up by mistake.
The Eurasia Tunnel under the Bosporus has been a point of controversy due to the traffic guarantee in its contract, which will lead to Turkish taxpayers forking out 323 million ($60 million) over the next three years.
Some 17.5 million vehicles passed through the tunnel last year, less than the guaranteed 25.6 million.