This paper conducted the theoretical analysis on 3D-PSF constitution, 3D-PSF energy distribution of different defocus amount and radial amount of focal-plane of digital biological confocal microscopic imaging system at first. In the study of 3D-PSF energy distribution of biological microscopic imaging system, it proposed the concept of energy ratio, conducted simulation experiments and calculated the energy ratio values of three groups with different magnifications, finally established its energy ratio spatial distribution model. Results showed that, 3D-PSF axial and radial energy rapidly decays with the axial and radial increasing, and the greater the magnification, the faster the energy attenuation, the more concentrated the energy.