Abstract We report a highly-uniform nanocomposite of polyglutamic acid (PGA) and gold nanoparticles (AuNPs) electrodeposited on a recycled battery graphite electrode (BGE) for the detection of Nalbuphine (NB), a semi-synthetic opioid. The sensor was optimized and characterized morphologically (via scanning electron microscopy, atomic force microscopy, and energy dispersive X-ray analysis) and electrochemically (via cyclic voltammetry, differential pulse voltammetry, and electrochemical impedance spectroscopy). Under optimized conditions, the PGA/AuNPs/BGE revealed two linear ranges, 2.5×10-8 to1.0×10-6 M, and 2.0 × 10-6 to 1.0 × 10-4 M for Nalbuphine (NB), that is equivalent to 9.825×10-3 to 0.393 µg/mL and 0.786 to 39.30 µg/mL, with R2 = 0.995 and 0.994, respectively, and showed good catalytic activity for the determination of nalbuphine in the presence of tramadol and the oxidation potential of these opioid analgesic drugs were separated. The sensor was successfully applied for the detection of NB in its pharmaceutical formulations, spiked urine, and human plasma samples, without applying any sample pretreatment, at a recovery range of 99±0.03 to102±0.02% and thus, the developed can be considered as a promising approach for NB abuse testing in clinical and forensic agencies.