A simple poly(arginine) film-modified carbon nanotube paste electrode (PAMCNTPE) was prepared using cyclic voltammetry (CV). The devised sensor was subjected to field-emission scanning electron microscope (FESEM) and CV characterization. The sensing of 0.1 mM pyridoxine (PY) was upgraded at PAMCNTPE as compared to the bare carbon nanotube paste electrode (BCNTPE). The PAMCNTPE detects the 0.1 mM PY at a specific potential 0.727 V with a current response of 10.68 µA. In the case of BCNTPE, the PY appeared at 0.798 V with a current 2.90 µA. The proposed analytical method was optimized by prime parameters such as the impact scan rate, pH and PY concentration. Under optimal conditions, the concentration of PY is directly proportional to oxidation current (Ipa) in linear range 2–10 µM, and 10–80 µM with a detection limit (LOD) of 9.6 × 10−7 M and limit of quantification (LOQ) of 3.21 × 10−6 M. The simultaneous determination, concentration variation analysis of PY is performed with riboflavin (RF) and interference analysis in detecting PY also examined. The proposed sensor was effectively applied for the determination of PY in natural food supplement with excellent recovery.