A new chiral biosensor fabricated by immobilizing bovine serum albumin (BSA) on gold-nanoparticle-modified glassy carbon electrodes, which could discriminate and detect phenylalanine (Phe) enantiomers, was proposed for the first time. The enantioselectivity was characterized by using scanning electron microscopy, cyclic voltammetry, electrochemical impedance spectroscopy and square wave voltammetry. The results showed that the BSA-modified electrode could enantioselectively recognize Phe enantiomers, and larger response signals were obtained from L-Phe. The factors influencing the performance of the biosensor were investigated. The enantiomeric composition of the L-/D-Phe mixture could be determined from the calibration curves. The modified electrodes have the advantages of simple operation, rapid detection and low cost.