To meet the performance test for the confocal laser scanning microscope (CLSM) accurately in some specific application occasions, the spatial resolution imaging principle of confocal laser scanning microscope was analyzed theoretically. And the micron line spacing as the measurement standard has tried to investigate the lateral resolution of CLSM in experiment. The value range of the lateral resolution was calculated by the fluctuation state of the output light intensity signal when there is the lateral movement between the objective with sample. At the same time, some reasons for spatial resolution are also been evaluated in theory. Experiments demonstrate that if the value of the line spacing standard is closer to the spatial resolution of CLSM, the standard can be utilized to test the spatial resolution. So we can use a a series of lines spacing standards with different lines spacing values to test the serial effective resolution. And in our experiment, we only measured line spacing standard with 8μm line width and 100μm line pitch with many times by CLSM, and the spatial resolution of the CLSM is obtained about more minimal than 0.3μm by the scanning curves.