Objective The aim is to explore an effective method of preparing the whole heart decellularized bio-derived scaffolds (HDBS) in rats for the heart tissue engineering. Methods The hearts were harvested from 30 adult SD rats. HDBS were prepared by the treatment with freeze thawing, typsin, SDS and Triton X-100, and obeserved the decellularization process at the same time. After decellularization, the scaffolds were examined through genomic DNA content analysis, HE staining, scanning electron microscope, transmission electron microscope and immunofluorescence. Results HDBSs maintained the three-dimensional structure with transparent appearance. Based on the DNA quantitative analysis, DNA content of the HDBSs was less than 3% compared to that of the control hearts. HE staining, scanning electron microscope and transmission electron microscope revealed that HDBS were decellularized completely and preserved the full extracellular matrix. Immunofluorescence results showed that collagen, elastin and other extracellular components were preserved completely without obvious nuclear components. Conclusion The HDBS prepared by the treatment with freeze thawing, typsin, SDS and Triton X-100 are decellularized completely, with the full extracellular matrix preserved, which are the ideal three-dimensional bio-derived scaffold material of the heart.