AIM: To explore an experimental method for primary culture of retinal neurons of neonatal rat. METHODS: Retina of postnatal 1-3 days SD rats was dissected into cell suspension by using trypsin digestion and cultured in vitro with DMEM/F12. Immunocytochemical methods were used to identify the cultured neurons. RESULTS: All cultured cells underwent adherence and some possessed axons，of which some were connected with each other．Most cells were neuron specific enolase(NSE)-positive detected by immunohistochemistry． CONCLUSION: Successful culture of retinal neuron cells in vitro is helpful in the research of retinal diseases.