The recent investigation targets to use adapted carbon paste (CP) with copper nanoparticles (CuNs) operating in a phosphate buffer (PBS) medium with a pH range of 5.0-8.0, to synthesize a novel, susceptible, and simple electrochemical sensor for the detection of one of the most important drugs, vitamin B. Copper (Cu) is one of the most three common essential trace elements found in the bodies of both humans and animals, along with iron and zinc for all crucial physiological and biochemical functions. Its properties, which are assessed using a variety of methods including scanning electron microscopy (SEM), cyclic voltammetry (CV), differential pulse voltammetry (DPV), and electrochemical impedance spectroscopy (EIS), have also drawn a lot of attention recently. We considered the effects of pH, buffer, scan rate, interference, and calibration curve. The susceptible electrode's linear calibration curve encompassed concentration values between 8.88 and 1000.0 µM. The calculated limits of detection and quantification were 32.12 and 107.0 µM, respectively. Furthermore, this method was established in real human urine samples and drug validation which have been shown satisfactory results for vitamin B detection.