A new electrochemical biosensor based on the human serum albumin/graphene oxide/3-aminopropyltriethoxysilane modified indium tin oxide electrode (ITO/APTES/GO/HSA) has been developed for the discrimination of tryptophan (Trp) enantiomers. The electrode has been characterized by scanning electron microscopy (SEM) and electrochemical techniques. The electrochemical behaviors of the enantiomeric pairs (d- and l-Trp) at the ITO/APTES/GO/HSA electrode have been investigated by cyclic voltammetry in the concentration range of 0.10-1.0. mM. A clear separation between the oxidation peak potentials of d- and l-Trp, at 0.86 and 1.26. V, respectively, has suggested that the ITO/APTES/GO/HSA electrode can be used as an electrochemical biosensor for the discrimination of Trp enantiomers. In order to find the percentage of an enantiomeric form of tryptophan in a mixture, the ITO/APTES/GO/HSA electrode is used for the simultaneous detection of d- and l-Trp which showed that the percentage of one enantiomeric form can be easily measured in the presence of the other. © 2012 Elsevier B.V.