The aim of this study was to investigate the optimal conditions for trypsinization in the culture of cerebral cortical neurons. Methods: The cerebral cortical region was dissected out from newborn rats,and then was treated with different concentration of trypsin at different time,in which the effects of trypsinization on cell viability were compared by cell morphology and cell count.Results: The cortical neurons with higher production and viability were obtained under the trypsinization condition of a final concentration of 0.25% tripsin at 37℃ for 5 min.The results show that 0.25% tripsin at 37℃ for 5 min made more monolayer cells and improved the viability of neurons.