This study demonstrated the adsorption behavior of 3,3′,5,5′-tetramethylbenzidine (TMB) on graphene oxide (GO) and L-cysteine functionalized reduced graphene oxide (L-Cys-rGO) sheets for the first time. To investigate the adsorption phenomenon of TMB on GO and L-Cys-rGO sheets, the effect of different pH values, adsorbent concentrations, and adsorption times were explored. The abundant hydroxyl groups contained on the GO surface has played an eminent role in TMB adsorption. The presence of the −SH functional group in the L-Cys molecule facilitates the π−π* and hydrogen bonding interaction between the L-Cys-rGO surface and TMB which results in efficient adsorption. On the basis of this finding, L-Cys-rGO decorated with bimetallic Au−Ag nanoparticles (Au−Ag/L-Cys-rGO) is utilized as peroxidase mimic activity for the TMB oxidation in the presence of H 2 O 2 . The adsorption of TMB on the catalytic surface is essential for the formation of blue color oxidized product (oxTMB); moreover, in the presence of Cr(VI) ion, it promotes the decomposition of H 2 O 2 to •OH radicals which enhances the TMB oxidation rate. On the basis of this mechanism, the proposed colorimetric probe has been utilized for selective and sensitive detection of Cr(VI) ions in aqueous medium up to the lower concentration of 26.39 μM. This work establishes the presence of L-Cys on a graphene oxide surface (L-Cys-rGO) which controls the growth of bimetallic Au−Ag nanoparticles and also helps in the adsorption of TMB on the catalytic surface. Thus, bimetallic Au−Ag nanoparticles-decorated on L-Cys functionalized GO opens a new path for easy naked eye colorimetric detection of environmentally toxic metal ions in an aqueous medium providing bright prospects in environmental monitoring.