Prestressed concrete has been widely used in bridge engineering. In the service life of bridge structures， the prestress loss is inevitable. Therefore， an accurate identification of effective prestress is important to ensure the long-term safety and stable opera?tion of bridge. The paper investigates change mechanism of influence line due to prestress and establishes the governing equation of the prestressed beam influence line. Both analytical and numerical solution are presented and verified by numerical simulation results. The influence of prestressing tendon line shape on the Deflection Influence Line （DIL） is further studied， the governing equation of the prestressed beam influence line is simplified. As such， a prestress identification method combined with the actual influence line is proposed. A numerical simulation test of the vehicle-prestressed beam bridge is carried out to calculate the dynamic response and extract the influence line of the bridge. The results are used to verify the correctness and feasibility of the proposed prestress identification method using the actual influence line of the bridge.