Gelatin protein hydrolysates (GPH) was prepared by using gelatin extracted from the skin of croaker (Johnius sp.), by the application of proteolytic enzymes extracted from gastrointestinal (GI) tract of two freshwater fish species rohu (Labeo rohita) and catla (Catla catla). The resultant hydrolysates were designated as gelatin protein hydrolysate-rohu protease (GPH-RP) and gelatin protein hydrolysate-catla protease (GPH-CP). Both the gelatin hydrolysates were fractionated into different ranges of molecular weight < 1 kDa, 1–3 kDa, 3–5 kDa, 5–10 kDa and > 10 kDa using ultrafiltration (UF) membranes. The fraction 1–3 kDa of GPH-RP hydrolysate and the fraction < 1 kDa GPH-CP hydrolysate were showed most potent angiotensin I-converting enzyme inhibitory (ACE) activity, and were subjected to liquid chromatography–mass spectroscopy (LC–MS/MS) for peptide sequencing, Fourier transform infrared (FT-IR) spectroscopy, scanning electron microscopy (SEM) and amino acid analysis. The peptide sequence of gelatin hydrolysate fractions of GPH-RP (1–3 kDa) and GPH-CP (< 1 kDa), were identified as GLTGRPGDAGPQGK and GFPGER with relative molecular mass (M+H⁺) values of 1310.67 Da and 662.32 Da, respectively. FT-IR spectra in both fractions revealed random coil-structure and β-sheet. Microstructure of GPH fractions had crystalline, broken glass like structure, flake-like and wrinkled structure.