Abstract: In order to obtain the optimal reaction conditions of immobilized lipase NovozymTM 435-catalyzed methyl esterification of fatty acids (FFA) from rapeseed oil, Plackett-Burman experiment design was employed to screen the significant factors affecting the esterification rate, which including lipase dosage, temperature, rotating speed and reaction time. The results of steepest ascent path experiment, Box-Behnken experiment and response surface analysis showed that reaction temperature was the most significant factor affecting the esterification rate, while reaction time was the least significant factor. Also, the reaction temperature and the rotating speed had significant interaction. Under the optimal conditions given by response surface analysis, i.e. substrate molar ratio 1.25∶1 (methanol/fatty acid), lipase dosage 11.5 g/kg FFA, temperature 42.2℃ and rotating speed 199 r/min, the measured average esterification rate reached 95.67% after 6 h reaction, which was consistent well with the predicted value (96.52%).