Abstract: In order to eliminate effect of fluorescent bead diameter on measurement results in traditional microscopic point spread function measurement method and improve the measurement accuracy, 2-D equivalent concentration distribution of fluorescent beads was deduced based on theoretical simulation and least square fitting method. The imaging process of fluorescent beads was simulated. The relationships among fluorescent beads diameter, full width at half maximum (FWHM) of fluorescent beads intensity distribution and FWHM of system point spread function were analyzed by using the least square fitting method and residual error fitting method. Precise FWHM correction model of point spread function was obtained. The results show that the relative error of point spread function FWHM is about 1% when measuring system point spread function with 100nm fluorescent beads. The precise measurement of point spread function is realizable with the optimized model.