To establish a more scientific and simple method for the culturing of cerebral cortical neurons from newborn rats and observe the interventional effect of different concentrations of urokinase. Methods: The cerebral cortex of newborn Wistar rats was extracted and then digested by papaya enzyme (2 mg/ mL) and an appropriate amount of DNase I enzyme. Neurons were cultured with serum-free Neurobasal medium. After 6 days, the purity of neurons was identified by immunofluorescence. Neurobasal media with final urokinase concentrations of 5 000 U/mL, 8 000 U/mL, 10 000 U/mL, 15 000 U/mL, and 20 000 U/mL were prepared for media changes. Neurons were observed under microscope for changes due to intervention from various concentrations of urokinase. Results: After 6 days of culture, the neurons were well-differentiated with abundant cytoplasm, and neuronal dendrites and axons extended and overlapped into a dense network. Neurons purity was identified as 88.2% by immunofluorescence staining. After intervention with urokinase at a concentration of 5 000 to 10 000 U/mL, no significant change in the culture system was observed under microscope within 2 h, but over time, a small number of neuronal cell disruption and network structure decrease could be observed with 10 000 U/mL at 4 h. With 15 000 U/mL and 20 000 U/mL, at 2 h after the intervention, fragmented material was observed at the bottom of the medium, and the intercellular network structure disappeared. Conclusion: In this study, a simple and efficient method of culturing newborn rat cortical neurons was established. When urokinase concentration was between 5 000 and 10 000 U/mL, it was safe to culture neurons within 2 h in vitro.