A chiral platform was designed for enantioselective recognition of naproxen (Nap) enantiomers using BSA biofunctionalized nanosheets of Toluidin blue O (TBO) and reduced graphene oxide (rGO) on the glassy carbon electrode. The novel TBO@rGO nanosheets acting as a supporter for the chiral selector (BSA) and electrochemical signal indicator had plenty of advantages, which were presented and explored via ultraviolet-visible (UV-vis) spectroscopy, scanning electron microscope (SEM) and electrochemical method. Kinetics showed the chiral platform had good redox signal and fast electron transfer. Under optimal conditions, much larger signal discrepancy was obtained after the chiral interface interacted with R-Nap, and a linear electrochemical response to Nap enantiomers was obtained from 5.0 x 10(-4) mol L-1 to 5.0 x 10(-3) mol L-1. with a low detection limit of 3.3 x 10(-7) mol L-1. Meanwhile, quartz crystal microbalance (QCM) for the interactions between BSA and Nap enantiomers further confirmed that R-Nap had a higher affinity with BSA. Furthermore, this simple chiral biosensor with excellent sensitivity and high stability is very expected to have more application in enantioselective recognition. (C) 2014 The Electrochemical Society. All rights reserved.