The study of interactions between proteins and surfactants is of relevance in a diverse range of applications including food, enzymatic detergent formulation, and drug delivery. In spite of sodium dodecyl sulfate (SDS)-induced unfolding has been studied in detail at the protein level, deciphering the conformation-activity relationship of a recombinant γ-glutamyltranspeptidase (BlrGGT) from Bacillus licheniformis remains important to understand how the transpeptidase activity is related to its conformation. In this study, we examined the enzyme catalysis and conformational transition of BlrGGT in the presence of SDS. Enzymatic assays showed that the transpeptidase activity of BlrGGT was greatly affected by SDS in a concentration-dependent manner with approximately 90% inactivation at 6 mM. Native polyacrylamide gel electrophoresis of SDS-treated samples clearly revealed that the heterodimeric enzyme was apparently dissociated into two different subunits at concentrations above 2 mM. The study of enzyme kinetics showed that SDS can act as a mixed-type inhibitor to reduce the catalytic efficiency of BlrGGT. Moreover, the t1/2 value of the enzyme at 55 °C was greatly reduced from 495.1 min to 7.4 min in the presence of 1 mM SDS. The I3/I1 ratio of pyrene excimer fluorescence emission changed around 3.7 mM SDS in the absence of BlrGGT and the inflection point of enzyme samples was reduced to less than 2.7 mM. The Far-UV CD spectrum of the native enzyme had two negative peaks at 208 and 222 nm, respectively; however, both negative peaks increased in magnitude with increasing SDS concentration and reached maximal values at above 4.0 mM. The intrinsic fluorescence spectra of tryptophan further demonstrated that the SDS-induced enzyme conformational transition occurred at approximately 5.1 mM. Tween 20 significantly suppressed the interaction of BlrGGT with SDS by forming mixed micelles at a molar ratio of 1.0. Taken together, this study definitely promotes our better understanding of the relationship between the conformation and catalysis of BlrGGT.