Objective To optimize the primary culture method for hippocampal neurons. Methods Hippocampus was harvested from fetal rats. They were obtained by enzymatic digestion and divided into groups A and B according to different times for replacing serum-free medium (4h after seeding in group A and 12h in group B). The cell growth was recorded on the 2nd, 3rd, 4th, and 5th days. Fluorescence assay was used to identify the cells with neuron specific enolization and determine the purity on the 7th day. Results After 4?6h, the cells attached to the wall. The cell body was enlarged on 2nd day. The nervous process erupted on 3rd day and extended into reticulation on 5th day. The fluorescence assay identified the neurons and indicated that the purity of group A was obviously higher than group B (P＜0.05). Conclusion The improved technique can cultivate well-growth hippocampal neurons with higher purity.