To optimize the preparation of ACE inhibitory peptides from black soybean (Glycine max (L.) Merr.), three enzymatic methods were compared, with the technical conditions optimized by response surface analysis. The black soybean protein hydrolysate inhibited 70.38% of the ACE activity. The ACE inhibitory peptides were isolated from black soybean protein hydrolysates by macro-porous resin, ultrafiltration, and Sephadex G-15. Four fractions were obtained, with each fraction having some ACE inhibitory activity. Fraction III exhibited the highest activity of 90.78%, and a molecular weight range of 145–468 Da. The ACE inhibitory peptides were stable across a range of pH values (2–10), at temperatures <40 °C, and in the presence of metal ions (Ca2+, K+, Mg2+), but had little resistance to digestive enzymes. These results indicated that the ACE inhibitory peptides of black soybean are substrate-type inhibitory peptides. •Microwave-assisted Alcalase hydrolysis was used to prepare black soybean protein hydrolysate.•The hydrolysis parameters were optimized using response surface methodology.•The hydrolysates exhibited an excellent stability in a wide variety of conditions.•ACE inhibition peptides with the highest activity were obtained by purification.