Beef potentiator was prepared by beef qu, which was previously prepared by a novel synergistic fermentation method (BPSF). Three kinds of other beef potentiators were prepared by commercial enzymes (protamex, flavourzyme and papain, BPCEs), which were used as controls to investigate the physicochemical properties of BPSF. Results showed that BPSF possessed a higher protein recovery (70.99%) and degree of hydrolysis (DH, 51.39%) than BPCEs. The tests of molecular weight distribution and free amino acids (FAAs) indicated that the moderate molecular weight peptides (1–5 kDa) were dominant (70.07–74.43%) in all beef potentiators. The low molecular weight peptides (<1 kDa), umami amino acids and total amino acids in BPSF, were 1.03- to 5.56-, 5.25- to 26.50- and 1.57- to 4.79-fold higher than those in BPCEs, respectively. Meanwhile, hierarchical cluster analysis indicated that BPSF was significantly different to BPCEs, and sensory evaluation showed that BPSF had apparently stronger umami taste than BPCEs.