System identification of structures is one of the important aspects of structural health monitoring. The accuracy and efficiency of identification results is affected severely by measurement noises, especially when the structure system is large, such as bridge structures, and when online system identification is required. In this paper, the least square estimation (LSE) method is used combined with the substructure approach for identifying structural parameters of a cable-stay bridge with large degree of freedoms online. Numerical analysis is carried out by first dividing the bridge structure into smaller substructures and then estimates the parameters of each substructure online using LSE method. Simulation results demonstrate that the proposed approach is capable of identifying structural parameters, however, the accuracy and efficiency of identification results depend highly on the noise sensitivities of loading region, loading pattern as well as element size.