Objective To culture three-dimensional contractible myocardial cell block of neonatal rats in atelocollagen scaffold and to further establish the foundation for heart tissue engineering. Methods After being cultured and purified, the neonatal rats’ cadiocytes were planted into atelocollagen scaffold for three-dimensional (3D) cultures. The growth of cadiocytes in atelocollagen scaffold was dynamically observed by inverted microscopy. Myocardial cell blocks in different periods were separately tested by HE staining, immunohistochemistry and immunofluorescence of myoglobulin, and observed under transmission electron microscope. Results After being planted, cadiocytes began to grow on atelocollagen scaffold in 6 hours, appeared mutual integration of myocytes and formed a complex with the scaffold to pulsate together on the 2nd day. And then, mutual integration among cadiocytes binding became more closely than before on the 6th day, and cadiocytes emerged into reticulation in the scaffold meshes on the 10th day. On day 20 of the culture,cadiocytes filled most of atelocollagen scaffold meshes and formed a tight block of myocardial tissue. The cadiocytes were always kept well the contractility and autorhythmicity in the whole course of culture. The growth cells in scaffold meshes were mainly cardiomyocyte, identified by morphological methods, with very few fiberlike cell. Conclusion Cadiocyte cultured in atelocollagen scaffold could develop the ideal and three-dimensional myocardial cell block with systolic function.