To reduce the side effects in the medication of Parkinson and Asthma, pyridoxine (PY) is administered along with L-3,4-dihydroxyphenyl alanine (L-dopa) and theophylline (TP), respectively. However, excessive dosage of PY leads to nervous disorder. Thus, a sensitive and selective electrochemical method was developed for the determination of PY in the presence of major interferences including TP, L-dopa, ascorbic acid (AA) and riboflavin (RB) using electrochemically reduced graphene oxide (ERGO) film modified glassy carbon electrode (GCE) in this paper. The ERGO fabrication process involves the nucleophilic substitution of graphene oxide at basic pH on amine terminal of 1,6-hexadiamine which was pre-assembled on GCE followed by electrochemical reduction. The electrocatalytic activity of the ERGO modified electrode was examined towards the oxidation of PY. It greatly enhanced the oxidation current of PY in contrast to bare and GO modified GCEs due to facile electron transfer besides pi-pi interaction between ERGO film and PY. Since TP and L-dopa drugs antagonize the drug action of PY, ERGO modified GCE was also used for the simultaneous determination of PY and L-dopa and PY and TP. Further, the selective determination of PY in the presence of other water soluble vitamins such as ascorbic acid and riboflavin was also demonstrated. Using amperometry, detection of 100 nM PY was achieved and the detection limit was found to be 5.6 x 10(-8) M (S/N = 3). The practical application of the present method was demonstrated by determining the concentration of PY in human blood serum and commercial drugs. (C) 2016 Elsevier Inc. All rights reserved.