The present invention relates to separation reagents and separation methods for mammalian cardiomyocytes, particularly human cardiomyocytes. Specifically, the present invention relates to a method for separating cardiomyocytes, which includes the following steps: a pretreatment step, a cleaning and decalcification step, a digestion step, an equilibration step, and a drip recalcification step, wherein at least An operating fluid contains BLEB and / or PAB or a physiologically acceptable salt thereof. Therefore, the present invention can significantly reduce the operation time of cardiomyocyte separation, and at the same time improve the viability and quality of the separated cells.