The characteristics of damped free vibration of concrete girder bridge under vehicle-running experiment are investigated. A method for damping estimation of structural damped free vibration by segment spectrum analysis is presented to improve the accuracy of damping identification. Firstly, the damped free vibration signal is intercepted by segmentation. Secondly, the amplitude of adjacent segments after FFT transform is taken to estimate the damping ratio. The accuracy of the present method and the traditional half power point method are compared by simulation. The results show that this method has higher accuracy than the half power point method when the length of the damped free vibration signal is limited. The method is used to estimate the damping ratio of a concrete continuous rigid frame bridge based on damped free vibration under vehicle running experiment. The first and second order vertical flexural frequencies and damping ratios are got. The results are credible and can be taken as the real damping ratios of the bridge.