Chinese pond turtle muscle peptide's molecular features, purification, structural characteristics, and antioxidant activity were investigated. The Flavourzyme hydrolysate demonstrated greater relative crystallinity (37.53%) than other hydrolysates using X-ray diffraction. The fourier transform infrared spectroscopy spectral changes, the second derivative spectroscopy in the amide-I region (1620–1650 cm⁻¹). Zeta-potential measurement was used to determine the surface charge ranging from − 32.73 to − 28.23 mV. Trypsin hydrolysate obtained the highest solubility (98.72% at pH 1.0) and emulsifying activity (182.81 m² g⁻¹ at pH 7.0), respectively. The Flavourzyme hydrolysate was separated by Sephadex G-10 filtration column chromatography, and three fractions (FH-1, FH-2, and FH-3) were obtained. The molecular weight was < 150 Da in fractions FH-3, FH-2, and FH-1, which were 93.25%, 85.22%, and 76.76%, respectively. The antioxidant activity showed the highest DPPH activity (71.32%) at 7 mg/mL in Fraction FH-2. SEM had a different shape (ball-drop) at FH-2 than the fractions protein (FH-1 and FH-3).