In the seismic response analysis of long span bridges, an optimal analysis method is proposed to solve Rayleigh damping coefficients. In the method, the optimization objective function is errors of structural displacement peaks. Meanwhile, considering that the fundamental-mode contribution is usually significantly larger than others, the modal damping ratio of fundamental frequency is equal to the exact value as constraint condition. Therefore, a constraint optimization equation is developed for solving Rayleigh damping coefficients. Then, the effects of both and optimal calculation modal numbers and reference degrees of freedom on the evaluation of Rayleigh damping coefficients were investigated with an 840m-long cable-stayed bridge. Comparing with the traditional Rayleigh damping, the errors of structural displacements and internal forces under four earthquake excitations were discussed. The numerical results show that the proposed method can reduce the calculation errors of displacements and internal forces effectively, and avoid artificial selection of two reference frequencies, which is suitable for calculation and analysis of engineering structures.