Reinforced concrete (RC) beams strengthened with prestressed fiber-reinforced polymer (FRP) laminates has been proved to be a rather effective strengthening technique in the field of bridge engineering. However, debonding failure usually occurs at the end of FRP in the strengthened beams on releasing the prestress due to the high interfacial shear stress. Analytical method to calculate the interfacial stress is developed in this paper. Through the establishment of mathematical model for the interfacial shear stress, the distribution of the interfacial shear stress and the longitudinal stress of FRP are presented explicitly in an analytical way. Moreover, the maximum prestress level is estimated to prevent debonding failure on releasing the prestress. Finally, experimental results of eight strengthened beams validate the analytical solution for the FRP longitudinal stress.