Objective To investigate the biocompatibility between the atelocollagen scaffold and 3D-cultured myocardial cells，and to find out the condition and an optimal biomaterial scaffold that can be applied to 3D-culture myocardial tissue. Methods The primary cultured myocardial cells were purified and then inoculated into the atelocollagen scaffold. The cells in the atelocollagen scaffold were observed by light microscope, scanning electron microscope(SEM) and transmission electron microscope (TEM) at different times (8d, 16d, 20d). Results On the first day, cardiac cells pulsating together with the atelocollagen scaffold could be detected under the microscope,which were pulsating complex. A plenty of cells in the atelocollagen mesh were observed under the light microscope, and the cells coalesced with the scaffold. The cells were compacted to the scaffold and coalesced with it at three stages under TEM. Those cells were sticked to the atelocollagen scaffold and expanded sufficiently under SEM. On the atelocollagen scaffold surface, these cells coalesced with lamellar.Conclusion The biocompatibility of the atelocollagen scaffold is better for cultured cardiac myocyte. It can be used as a natural material for 3D-cultured cells, and is suitable for 3D-cultured cardiac cells and cardiac tissues.