Aim To explore a novel experimental method for detecting cardiomyocyte proliferation at the live cell level, and to exclude false positive cells caused by multinucleate cardiomyocytes, so as to provide experimental reference for accurate detection of cardiomyocytes proliferation. Methods Use the cell division cyclin 20 (CDC20) and spastic paraplegia 20 (SPG20) as the molecular beacons targets. These molecular beacons allow for CDC20 and SPG20 detection in live cells, through Lipo-2000 transfection. The double positive cells are the proliferating ones that undergo cytokinesis. Results When the cells did not proliferate, the probes could not detect the target sequences and the cells cannot be detected by the fluorescence. When the nuclei replicated and divided without cytokinesis, the sequences could not be detected by both two probes at the same time, and the cells also could not be detected. When cells underwent mitosis with complete cytokinesis proliferation, both two fluorescence probes could be detected simultaneously. Conclusion The molecular beacons-based approach targeting CDC20 and SPG20 identified a proliferative subset of cardiomyocytes, which could exclude false positive problems of cell proliferation caused by nuclear proliferation and improve the accuracy of cell proliferation detection.