The effects of two-step enzymolysis of corn yellow powder on the preparation of high fischer ratio (F value) oligopeptides were studied. The results showed that the protease type, enzyme addition amount, solid-liquid ratio, pH, enzymatic temperature and reaction time had certain influences on the protein conversion rate and F value of active peptides. The primary enzymolysis conditions obtained by the single factor and orthogonal test optimization were as follows:alkaline protease amount 12.5kU/g (based on corn yellow powder mass), pH 9.0, solid-liquid ratio 1∶35g/mL, enzymatic temperature 50℃, and reaction time 3.0h. The protein conversion rate reached 85.61%. Using first-step enzymatic solution as raw material, the conditions of secondary enzymolysis optimized by the single factor and orthogonal test were obtained as follows:complex flavor protease amount 2.7kU/mL (based on volume of the first-step enzymatic solution), pH 7.0, enzymatic temperature 45℃, and reaction time 2.5h. After two-step enzymolysis of corn yellow powder, the F value of the samples adsorbed by activated carbon reached 28.30, and the oligopeptide yield was 35%. The molecular weight of oligopeptides were between 493.46-912.84Da and high fischer ratio oligopeptides made up more than 85%. It provides theoretical and technical guidance for the comprehensive utilization of corn yellow powder resources.