Copper oxide nanoparticles (CuO Nps) were synthesized using Caesalpinia bonducella seed extract via a green synthetic pathway and were evaluated for electrocatalytic properties by carrying out electrochemical detection of riboflavin [vitamin B 2 (VB 2 )]. The seeds of C. bonducella are known to have strong antioxidant properties arising due to the presence of various components, including citrulline, phytosterinin, β-carotene, and flavonoids, which serve as reducing, stabilizing, and capping agents. The synthesized CuO Nps were characterized using UV−visible spectroscopy, Fourier transform infrared spectroscopy, thermogravimetrc analysis−differential thermal analysis, X-ray diffraction spectroscopy, X-ray photoelectron spectroscopy, and scanning electron microscopy and further used as a modifier for a graphite electrode surface. The modified electrode was electrochemically characterized by cyclic voltammetry, square-wave voltammetry, and chronoamperometry techniques and then assessed for electrocatalysis by carrying out the detection of VB 2 . The electrochemical sensor could be used for nanomolar detection of VB 2 with an observed linear range of 3.13−56.3 nM with a limit of detection of 1.04 nM. The electrode showed good stability and reproducibility over a period of 120 days. The CuO Nps were further analyzed for antibacterial effect with Gram-positive and Gram-negative bacteria, and in both cases, high antibacterial activity was clearly observed. The newly synthesized nanoparticles, thus, proved to be an interesting material for electrochemical and biological studies.