The determination method of polymer micro sphere’s particle size used in profile control by laser particle size analyzer was established. The sample preparation method was optimized in several aspects，such as dispersant，loading mode，stirring speed and stirring time. The optimum dispersing condition was obtained as follows: one drop of sample was added in 50 g petroleum ether with stirring speed of 400 r/min and stirring time of 1 min. This method had good reproducibility with the relative standard deviation of 0.0591%. Using LA-950S laser particle size analyzer under the optimum condition，the particle size of micro sphere was 1.8592 μm by volume base and 308.79 nm by number base. Therefore，the evaluation method should be chosen according to the objective.