The anti-exercise-fatigue effect of active peptides derived from fish nicotinamide adenine dinucleotide Dehydrogenase (NADH) was investigated by simulating the hydrolysis of NADH Dehydrogenase protein subunits in five common fish species in China using computer simulations. The oligopeptides obtained through simulated hydrolysis were docked with lactate Dehydrogenase (LDH) and creatine kinase (CK) to identify the oligopeptides with ultra-high affinity for both enzymes (docking fraction ≤ -160), whereas Pymol and Ligplot+ software were used to display and analyze the mechanisms of interaction for molecular docking. The results showed that a total of 72 oligopeptides with ultra-high affinity were obtained from seven different NADH Dehydrogenase protein subunits of five fish species after computer-simulated hydrolysis and molecular docking. There were 36 oligopeptides with ultra-high affinity for LDH and CK, among which the oligopeptide with the highest docking score and the most frequent occurrence was PTIW (-198.762, -204.400). These results establish a theoretical foundation for improving the high-value utilization of fish protein and developing functional foods for anti-exercise-fatigue.