A modified precipitation method was used to prepare poly(lactic acid) (PLA) nanoparticles, and the response surface method applied to optimize the preparation parameters. The results showed that there were three main factors affecting the formation of nanoparticles in the preparation process, amount of PLA, volume fraction of ethanol in oil phase and total volume of oil phase. The mathematical model was established using the response surface methodology, and the optimal conditions were obtained with the aim of minimum average particle size, which were the amount of PLA at 76.82 mg, volume fraction of ethanol in oil phase 40%, and oil phase volume 19.49 mL. The size of PLA nanoparticles prepared under the optimal conditions was 109.1 nm and the PDI value 0.084, which was closer to the predicted value. The PLA nanoparticles were spherical solid particles with good dispersibility and uniform size distribution. The PLA nanoparticles could be phagocytosed by macrophage, and used in later immune experiments.