In the hydrolysis of yeast protein, exoprotease dosage strongly affected the recovery of protein and DH and sensory characteristics. The combined treatment of endoprotease and exoprotease (0.6% Protamex and 0.6% Flavourzyme) resulted in high solid recovery (48.3–53.1%) and the best flavor profile. Among various treatment sequences using multiple enzymes, the treatment with protease followed by nuclease was desirable for yeast hydrolysis in terms of 5′-GMP content and solid and protein recovery. The optimal concentrations of both 5′-phosphodiesterase and AMP-deaminase were found to be 0.03%. The treatments using optimized conditions produced yeast extract that contained mostly low molecular weight peptides and free amino acids, with a high solid recovery of 55.1%. Consequently, a three-step process using four enzymes was developed for the production of yeast extract utilizing brewer's yeast.