In the present study, a novel approach was designed to construct a reagentless chiral biosensor for highly selective recognition of N-isobutyryl-cysteine enantiomers. The reduced-graphene oxide sheets (rGO) and thionine (TH) were firstly stepwisely synthesized onto modified glassy carbon electrode using glutaraldehyde as a bifunctional reagent, and gamma-globulin was adsorbed on the electrode surface as a chiral selector. TH-rGO with excellent redox electrochemical activities brought in more redox probes for chiral recogniton, which could improve the sensitivity of the biosensor. Good reproducibility and sufficient stability were obtained with the advantages of TH-rGO and glutaraldehyde reagent. Moreover, the proposed biosensor showed an obviously decreased current peak after adsorption of N-isobutyryl-L-cysteine isomer. As compared with penicillamine or cysteine enantiomers, the current biosensor showed good enantioselectivity for sensing N-isobutyryl-cysteine enantiomers. (C) 2013 The Electrochemical Society.