Objective To establish a stable and fast method for primary culture of mouse cardiomyocytes. Methods Dishes were coated with polylysine firstly. A two-step approach was used to isolate and digest mouse cardiomyocytes cells (0.25% trypsin in 4℃ overnight and 0.5 mg/mL to 1.0 mg/mL collagenase +5 mg/mL albumin collagen digestion liquid in 37℃ for short-time digestion), then the cardiomyocytes were purified through differential adhesion for 70 min and 5-bromodeoxyuridine (BrdU). The cell morphology was observed under an inverted microscope. The survival rate of cardiacmyocytes was detected by trypan-blue staining and their purity was identified by α-actinin immunofluorescence staining. Results The cardiomyocytes were in good shape and pulsed spontaneously. The survival rate of the cardiomyocytes reached 98% and the purity was 95%. Conclusions This method described in this study is an ideal method for primary culture of mouse cardiomyocytes with a high survival rate and high purity.