Objective To compare three methods for culture of fetal cortical neurons of SD rats and find out the suitable culture conditions of fetal cortical neurons in vitro.Methods The cortex of 16-18-day embryonic rat was used for culture in this study.Mechanical dissociation, trypsin digestion and papain digestion were applied respectively to the neuron culture.The morphological characteristics of neuronal cells at different time points were observed and neuron purity was identified by immunofluorescence staining assay.Results High purity of the fetal rat cortical neurons was successfully achieved by all the three culture methods, and each had distinct morphological characteristics at different time points.The purity of neurons was 96.28%, 95.63%and 97.34%, respectively, with no significant differences among the three groups (P>0.05).Conclusions The three culture methods are improved in our study.Stable neurons with high purity can be obtained by all the three methods respectively, and each of these methods has distinct characteristics.