Objective To culture neonatal rat ventricular myocytes after enzyme digestion, explore method to obtain myocardial cells with high survival rate and high purity, and offer cell model for physiological and pharmacological study. Methods Neonatal rat ventricular tissues were digested by trypsinase and collagenaseⅠ, the digestion time and temperature were controlled. Myocardial cells were purified by adherence separation method and chemical inhibition method, and were cultured in vitro. Morphocytology changes were observed by fluorescence microscopy, troponinⅠ was determined by flow cytometry, and free Ca²⁺  in cytoplasm of cardiomyocytes was detected with Fluo-3/AM. Results Cardiomyocytes began to adhere and grow after 4 h, few individual cells began to beat spontaneously after 24 h, and cardiomyocytes got together and beat synchronously in clusters gradually, with the cellular shape and structure being intact. The positive rate of troponinⅠ was 91%, Ca²⁺ staning was positive in cytoplasm of all cardiomyocytes, and periodic variation was detected along with cell pulsation. Conclusion A large number of vigorous cardiomyocytes with high purity can be obtained by enzyme digestion method, which may provide favourable model for cardiovascular research.