Based on modal perturbation method and mode superposition method, a calculation method for vertical seismic response analysis of prestressed Bridges is established. Through the analysis of two typical engineering examples, several problems in vertical seismic response analysis of Bridges are discussed. The results show that: (1) The preloading increases the natural vibration frequency of the bridge, and the influence is mainly reflected in the low-order natural vibration frequency. With the increase of the mode order, the influence of the preloading decreases. For prestressed Bridges with 20m and 32m spans, the vertical fundamental frequency increases by 5.3% and 6.3%, respectively, while the fifth vertical frequency increases by only about 1%. (2) The bending moment of the mid-span section of the prestressed beam is reduced by about 30% and 25% under the action of the surface wave, and the earthquake resistance of the bridge is improved by the prestressing force. (3) The contribution of higher order modes in vertical seismic response can be ignored. (4) The vertical seismic effect accounts for 24.56% and 26.15% of the load combination effects of the mid-span bending moment of prestressed Bridges at 7 degrees of fortification intensity, and 38.93% and 41.46% at 8 degrees of fortification intensity, respectively, indicating that the influence of vertical seismic response should be considered in the seismic design of prestressed Bridges.