This study aimed to characterize the substrate affinity and catalytic efficiency of bovine milk γ-glutamyltransferase (BoGGT) towards different donors and acceptors by comparing it with a reference (Bacillus amyloliquefaciens, BaGGT). Quantitation of γ-glutamyl peptides and free amino acids was conducted in combination with enzymatic kinetic. Kokumi peptides were generated from whey protein hydrolysates through transpeptidation catalyzed by both GGTs. BaGGT has a higher transpeptidase activity than BoGGT when γ-glutamyl-p-nitroanilide (γ-GpNA) or glutamine acts as a donor. Glutamine is a better γ-glutamyl donor than γ-GpNA for both GGTs. Furthermore, membrane-free BoGGT has a more efficient activity and higher substrate affinity than the native BoGGT. BoGGT has the highest affinity with Val-Gly and can produce γ-Glu-Val-Gly, a substance with a strong kokumi intensity and the lowest taste threshold. This study reveals that the catalytic ability of GGT is highly dependent on the acceptor, and membrane interactions restrict the transpeptidase activity of BoGGT.