β-Glucosidases (BGL) are key members of the cellulase enzyme complex that determine efficiency of lignocellulosic biomass degradation, which have shown great functional importance to many biotechnological systems. A previous reported BGL from Neosartorya fischeri (NfBGL) showed much higher activity than other BGLs. Screening the important residues based on sequence alignment, analyzing a homology model, and subsequent alteration of individually screened residues by site-directed mutagenesis were carried out to investigate the molecular determinants of the enzyme's high catalytic efficiency. Tyr320, located in the wild-type NfBGL substrate-binding pocket was identified as crucial to the catalytic function of NfBGL. The replacement of Tyr320 with aromatic amino acids did not significantly alter the catalytic efficiency towards p-nitrophenyl β-D-glucopyranoside (pNPG). However, mutants with charged and hydrophilic amino acids showed almost no activity towards pNPG. Computational studies suggested that an aromatic acid is required at position 320 in NfBGL to stabilize the enzyme-substrate complex formation. This knowledge on the mechanism of action of the molecular determinants can also help rational protein engineering of BGLs.