In a detection system of STR labeled by multiple fluorescence, the overlaps do occur between the different colored fluorophores owing to the band broadening characteristic of the employed fluorescence spectrum. It is crucial to obtain effective energy distribution specific for different wavelengths so as to improve the utilization efficiency of fluorescence intensity. To reach this goal in practice, we propose a data processing method based on matrix analysis. Firstly, a spectral calibration must be done for STR fluorescence spectral analysis, and then a emission spectral distribution of each colored fluorophore excitated by laser is created from the detection signal. And then, the matrix normalization is computed from the above fixed spectrum distribution. And lastly, the inversion of the correct matrix is used to get the resolved and desirable STR spectrogram. In addition, performance evaluation shows that this method based on matrix analysis can make use of the fluorescence spectrum energy of each wavelength and resolve the overlapping spectrum effectively.