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This study investigates the enzyme-less biosensing property of the zinc oxide/carbon nano-onion (ZnO/CNO) nanocomposite coated on a glassy carbon electrode. The ZnO/CNO nanocomposite was synthesized using the ex situ mixing method, and the structural characterization was done using XRD, SEM, and TEM, whereas functional groups and optical characterization were done through FTIR and UV–visible spectroscopy. The electrochemical sensing response of the ZnO/CNO nanocomposite for the linear range of glucose concentration (0.1–15 mM) was examined using cyclic voltammetry (CV) with a potential window of −1.6 to +1.6 V using 0.1 M NaOH as an electrolyte. The ZnO/CNO nanocomposites showed enhanced sensing ability toward glucose with a sensitive value of 606.64 μA/mM cm2. Amperometric i-t measurement supports the finding of CV measurement and showed good sensing ability of the electrode ZnO/CNO nanocomposite material for up to 40 days. The enhanced electrocatalytic activity of the ZnO/CNO nanocomposite is explained due to the synergetic effect of both ZnO and CNO. Our findings suggest a high potential for ZnO/CNO nanocomposite-based glucose biosensors, which could be further utilized to develop noninvasive skin-attached sensors for biomedical applications.