This work describes the electrocatalytic properties of a carbon composite electrode (CCE) modified with Prussian blue (PB) nanoparticles (NPs) for the electrocatalytic oxidation of pyridoxine (PN). The morphology of the PBNP-modified CCE was characterized by scanning electron microscopy (SEM). The mechanism and kinetics of the catalytic oxidation reaction of PN were monitored by cyclic voltammetry and chronoamperometry. The rate-limiting step of the charge transfer reaction was found to be a one-electron abstraction. The value of α, k, and D were calculated as 0.66, 6.7×104 M−1 s−1, and 1.88×10−5 cm2 s−1, respectively. The modified electrode showed electrocatalytic activity toward the oxidation of PN and was used as an amperometric sensor. The sensor exhibited good linear response for PN over the concentration ranges 5–69 and 1–80μM with detection limits of 0.51 and 0.87μM, and sensitivities of 0.97 and 0.673AM−1 cm−2 in batch and flow conditions, respectively. Some important advantages such as simple preparation, fast response, good stability, and reproducibility of the sensor for the amperometric determination of PN were achieved.