The structural and physicochemical characteristics of protein hydrolysates prepared from Chinese sturgeon through the enzymatic hydrolysis process were evaluated. Two different enzymes including papain and alcalase 2.4L were used in the hydrolysis process. The papain enzyme significantly increased the degree of hydrolysis (20.62%) and decreased the ζ-potential (12.4 ± 1.31 mV) as compared to the alcalase enzyme, which represented 15.55% and 15.53 ± 0.77 mV, respectively. Alcalase 2.4L hydrolysate exhibited smaller particle size (822.047 ± 61.26 nm) than papain hydrolysate (1425.39 ± 44.82 nm). Hydrolysis by papain and alcalase 2.4L enzymes decreased the molecular weights (MW ≤ 1,000 Da) to 98.27% and 86.84%, respectively. The surface hydrophobicity and turbidity of the hydrolysates significantly affected by enzyme type and protein concentrations. By using the X-ray diffraction analysis, the papain hydrolysate showed a higher relative crystallinity degree (30.33%) than alcalase 2.4L hydrolysate (29.40%), whereas the Fourier transform infrared spectroscopy showed more clearly peaks for the amide bands of alcalase hydrolysate. The thermal properties also affected by enzymatic hydrolysis conditions, since the melting temperatures were 159.17 and 149.58 °C, whereas the rate of mass loss was 67.04% and 62.8%, for papain and alcalase hydrolysates, respectively. PRACTICAL APPLICATION: The enzymatic hydrolysis process of proteins is employed to obtain the nutritionally and functionality important peptides that result during the preparation of fish protein hydrolysate. The objective of this study was to investigate the structural and physicochemical characteristics of protein hydrolysate prepared from Chinese sturgeon. This study showed that these characteristics were affected by enzymatic hydrolysis conditions especially enzyme type. The finding of this study may be useful in terms of providing new information on the properties of the protein hydrolysate and the structural changes resulting from controlled enzymatic hydrolysis conditions. Overall, these conditions could potentially alter the secondary structure of the protein hydrolysates or peptides and enhance their functional properties.