BACKGROUND Deer heart is a valuable traditional Chinese medicinal material. At present, there is no functional component or symbol component for deer heart, and it is difficult to identify them by physical and chemical methods. In this study, we established deer heart identification method based on mtDNA cytb gene, and developed a deer heart DNA detection kit. RESULTS There were specific amplification bands in reference medicinal materials and true deer hearts at 194 bp, but no amplification bands in negative control and blank control. Deer heart DNA detection kit has good specificity, reproducibility and stability, and its sensitivity can reach 0.5 mg. CONCLUSIONS The application of the deer heart DNA detection kit is simple and easy to operate, and the results obtained using the kit is stable, which is suitable for popularization and application