In this study, nickel oxide nanoparticles (NiONPs) were produced by the co‐precipitation method and characterized by scanning electron microscopy, X‐ray diffraction (XRD), Fourier transform‐infrared and Ultraviolet‐visible spectroscopy. XRD analysis showed an average crystal size of 6 nm, while SEM analysis exemplified the oblong form with a particle size of 12 nm. As‐prepared NiONPs were combined with multiwalled carbon nanotubes (MWCNTs) and graphite powder to prepare NiONPs‐MWCNTs modified carbon paste electrode (MCPE). The fabricated NiONPs‐MWCNTsMCPE sensor was used to analyze Gemfibrozil (GEM) in pharmaceutical formulations. Electrochemical methods such as cyclic voltammetry, differential pulse voltammetry, and electrochemical impedance spectroscopy employed in sensor characterization and electroanalysis of GEM. The sensor exhibited excellent electrochemical behavior towards GEM. It showed a lower limit of detection (LOD, 2.7×10−8 M), the limit of quantification (LOQ, 9.07×10−8 M), higher sensitivity, higher linear range, and long‐term stability compared to the reported methods. The NiONPs‐MWCNTsMCPE sensor was evaluated for practical application by performing spiking tests in pharmaceutical formulations and human urine samples, demonstrating significant recovery, high effectiveness, and accuracy. Further, nanoparticles manifested promising antibacterial action against Staphylococcus aureus, E. coli, Klebsiella aerogenes, Pseudomonas aeruginosa, and Klebsiella aerogenes.