Aim To optimize the conditions and methods of SD rat cardiomyocytes and cardiac fibroblasts in vitro culture. Method Digested ventricular tissue with trypsin first, and then collagenaseⅡ. Collected and cultured neonatal rat cardiac myocytes and fibroblasts through the way of differential adhesion for two times. The basic shape change of cardiac myocytes and cardiac fibroblasts were observed under the opitical microscope. Cardiac myocytes were assayed by cardiac troponin immunofluorescenee and the second generation cardiac fibroblasts were assayed by vimentin immunofluorescence. Results Cardiomyocytes began to adhere and grow after 2～4 hours The adherent rate increased substantially and the cells beated spontaneously after 12～24 hours 48 ～72 hours later cardiomyocytes adhered to be clustered Both of the cells’ survival rate and purity reached more than 95%. The second generation to the fourth generation cardiac fibroblasts grew wel1 and its purity was as high as 98%. Conclusion A great quantity, purity and high survival rate of cardiocytes and cardiac fibroblasts can be effectively cultured by this method. And it provides an ideal experimental model for the cardiovascular disease and clinical studies.