Channa striatus is high-protein food with many health functions. This study aimed to prepare, screen and identify the angiotensin-converting enzyme inhibition peptides (ACEIPs) from C. striatus hydrolysates by response surface methodology and bioaffinity ultrafiltration coupled with LC-Orbitrap-MS/MS and molecular docking. The optimal conditions for preparing ACEIPs were hydrolysis temperature 55 °C, hydrolysis time 3 h, pH 9, solid–liquid ratio 1:20 g/mL, and enzyme addition 5%, the ACE inhibition and molecular weight distribution of obtained hydrolysate was 54.35% and 8770–160 Da, respectively. Seven novel ACEIPs were screened through the established high-throughput screening approach, among which, EYFR and LPGPGP showed the strongest ACE inhibition with the IC50 value of 179.2 and 186.3 μM, respectively (P > 0.05). Molecular docking revealed that three and ten hydrogen bonds were formed between ACE and LPGPGP and EYFR, respectively, S1 and S2 were the major active pockets, but the major driving forces varied.