Abstract: In order to obtain experimental 3-D point spread function of a digital confocal microscope optical system, a fluorescence microsphere was used to simulate the point light source, a digital confocal microscope optical system was used to collect the slice images of the fluorescent microsphere with different defocuses, and the multi-image averaging method was used to denoise the slice images. Experimental 3-D point spread function of the microscope optical system was constructed. 3-D microscopic image restoration was carried out by experimental point spread function and was compared with that of theoretical point spread function. The results show that both the point spread functions have good restoration effect for image restoration, but experimental 3-D point spread function obtained by correct experimental method is more consistent with optical transmission characteristics of an optical system and has more accurate recovery effect. Experimental 3-D point spread function constructed in the study provides a better choice for the next step of biological microscopic image restoration. It provides an effective method for the construction of 3-D point spread function of digital confocal microscopes.