Objective To compare two separation medium of isolation of adult rat cardiomyocytes, and to observe the characteristics of excitation-contraction coupling of cardiomyocytes. Methods The isolated adult rat heart was hanged on to the Langendorff apparatus for aortic counter-current perfusion and collagenase digestion using two different separation medium. The single cardiomyocytes were cultured and infected with adenovirus. The morphological features of cardiomyocytes were observed with microscope and fluorescent microscope. The shortening-re-lengthening features of sarcomere and the intake-discharge features of calcium were simultaneously recorded by IonOptix equipment. Results 70% rod-shaped with clear-striation adult rat cardiomyocytes could be obtained with the stated two separation medium and cultured in serum-free medium for more than 7 days. GFP could express more than 7 days when the cardiomyocytes were infected with adenovirus. Cardiomyocytes obtained by the first separation medium could not contract with the electrical stimulation, while cardiomyoctyes obtained by the second separation medium could be used for the detection of excitation-contraction coupling. The shortening fraction of sarcomere was 11.61%±2.15% and the relaxing time was (0.177±0.031) s. The amplitude of calcium transient was 30.79%±9.74 % and the decaying time of calcium transient was (0.300±0.074) s. Conclusion With the stated two separation medium, adult rat cardiomyocytes can be well isolated, cultured and infected with adenovirus. The second separation medium can be used for the detection of excitation-contraction coupling characteristics.