In this study, a method for the sensitive, rapid, efficient and simultaneous determination of the riboflavin (VB2) and pyridoxine (VB6) by voltammetry technique was developed. For this purpose, a novel electrochemical sensor (44-DABP/GC) based on GC electrode modified with 4,4'-diamino benzophenone was fabricated by the cyclic voltammetry. Best analytical performance was obtained in 0.1 M, pH 4.5 acetate buffer solution, with a scanning rate of 100mVs−1, a puls time of 10ms, a scan increment of 10mV and a pulse amplitude of 125mV. 44-DABP/GC electrode exhibited good analytical performance for the determination of VB2 and VB6. Three linear ranges in wide concentration ranges for VB2 from 13nM to 660μM, and two linear ranges for VB6 from 15.7μM to 2210μM were obtained with the detection limit of 8.52nM and 4.70μM, respectively. Besides, repeatability, reproducibility and interference studies were performed. The proposed sensor was applied successfully in food and pharmacological samples.