A multi-input and multi-output unbiased frequency response function (MMUFRF) estimation is put forward based on the feature that the mean of the incorrelate signal CSD (Cross Spectral Density) tends to zero. The proposed method is realized by MATLAB programming. Two-input and four-output test is carried on using the dynamic signal test systems and its MMUFRF is acquired by MATLAB program. Meanwhile, numerical simulation of the frequency response functions is made by means of finite element method. The simulation results show that the proposed MMUFRF can correctly estimate the frequency response functions of real structures.