The Chinemys reevesii was selected as the raw material, and the inhibitory effect of the polypeptide of papain enzymatic hydrolysate on the proliferation of human breast cancer cell MCF-7 was identified as the activity detection index in this study to evaluate the cytotoxicity effects of extracted peptides by using MTT assay. The enzymatic hydrolysate was retained by ultrafiltration membrane with a molecular weight of 10 Ku first, and then, the active components were purified by sephadex G-75, sephadex G-50, and sephadex LH-60. The results of RP-HPLC spectrum indicated that a high-purity anticancer component, here named TP-1 was isolated and purified with an IC50 value of approximately 2.7 mg/mL, showing a significant dose-dependent and time-dependent anticancer effect, and little toxicity to normal cells. In addition, the physicochemical properties of TP-1 were characterized by ultraviolet, infrared and circular dichroism, and the molecular weight of the main component of the polypeptide was identified by mass spectrometry. The results manifested the existence of secondary structure of β-sheet, β-turn and random coil in TP-1, and the molecular weight of the principal component in TP-1 was about 1410.7 u. The research laid a solid foundation for further studies on anti-tumor mechanism of bioactive peptides from C. reevesii.