The measurement principle of fluorescence spectrum on Na2KSb film of multi-alkali photocathode was described and three samples were measured in the position of different radius. The data result shows that the peak wavelength of fluorescence spectrum on Na2KSb film from center to edge of the cathode surface gradually increases, and the peak fluorescence intensity gradually increased as well. The reason is that the antimony atom density of cathode surface from the center to the edge gradually reduces. When the antimony in Na2KSb film exceeds stoichiometry required, the fluorescence peak wavelength shifts towards the short-wave direction, while the fluorescence intensity decreased at the same time. When the antimony in Na2KSb film achieves stoichiometry required, the fluorescence peak wavelength reaches the maximum value, while the fluorescence intensity reaches the strongest at the same time. By fluorescence test one can judge whether the stoichiometry of Na2KSb film reaches to the ratio 2:1:1 or not, in another words whether antimony in Na2KSb film is overdose or not. In addition by measuring the fluorescence spectra at different positions of the cathode surface, we can measure component uniformity in the Na2KSb cathode film. The more uniform antimony atom density is in the cathode surface, the more accurate the monitor method of film growth by measuring changes of the cathode photocurrent is, thus component uniform can be better, Na2KSb film thickness can be thicker, long-wave absorption of visible light is more, the sensitivity of the cathode is higher. Therefore, during the manufacturing process of multi-alkali cathode of image intensifier, one has to make the uniform antimony atoms density on cathode window surface in order to achieve higher sensitivity.