This paper presents a new identification approach to prestress force. Firstly, a bridge deck is modeled as a prestressed Timoshenko beam. The time domain responses of the beam under sinusoidal excitation are studied based on modal superposition. The prestress force is then identified in the time domain by a system identification approach incorporating with the regularization of the solution. The orthogonal polynomial function is used to improve the noise effect and obtain the derivatives of modal responses of the bridge. Good identification results are obtained from only the first few measured modal data under both sinusoidal and impulsive excitations. It is shown that the proposed method is insensitive to the magnitude of force to be identified and can be successfully applied to indirectly identify the prestress force as well as other physical parameters, such as the flexural rigidity and shearing rigidity of a beam even under noisy environment.