A three-dimensional image of a living cell is helpful for cell secretion study. In this report, the three-dimensional fluorescence deconvolution microscopy for observing living cells was studied, because this technique can obtain a quick three-dimensional imaging with minimal fluorescence quenching and cytotoxicity for living cell observation. The property of three-dimensional point spread function (PSF) of imaging system was analyzed. The relationship between experimental and theoretical PSF was illustrated, and the theoretical PSF was proved that it could reflect the principle of imaging system with NA 1.65 objective in use. Three-dimensional deconvolution algorithm in this report was proved effective by well-defined three-dimensional specimens. Furthermore, the rat pancreatic beta cell secretory vesicles labeled by acridine orange was observed by using this technique. Results showed that the blurring induced by out-of-focus light was removed by the deconvolution algorithm effectively, under current experiment conditions (with NA 1.65 objective) the experimental PSF approximated the theoretical PSF very well, and deconvolved living cell images exhibited the spatial distribution of the secretory vesicles clearly.