In order to improve Qos quality of Dense Wavelength Division Multiplexing optical fiber transmission, a Qos unicast routing mechanism based on ant colony optimization algorithm is proposed. The algorithm establishes the main optical path and the spare optical path through the ant colony classification according to the dynamic optical path connection request, so as to minimize the blocking probability caused by the masterslave multiplexing technique. In addition, in order to solve the problem of local optimal solution, the ant colony algorithm is optimized by setting genetic factors. NS2 simulation results show that compared with the traditional routing algorithm, the proposed QoS unicast routing has better feasibility and global optimization ability, which can effectively reduce the blocking probability and improve the utilization of network resources.