In this study, we successfully developed a novel nanocomposite comprising hexagonal nickel oxide nanosheets (HNONS) and carboxyl-terminated reduced graphene oxide (Tr-rGO) using a hydrothermal process followed by calcinations for the detection of H2O2 and ascorbic acid. The surface morphology and structure of HNONS@Tr-rGO were characterized using field-emission scanning electron microscopy, X-ray photoelectron spectroscopy, and Raman spectroscopy. The electro catalytic activities of HNONS@Tr-rGO toward H2O2 and ascorbic acid were investigated using cyclic voltammetry, amperometry, and electrochemical impedance spectroscopy. Under optimized conditions, the HNONS@Tr-rGO modified electrode exhibited a high sensitivity of 177.83 μAmM−1cm−2, wide linear range from 0.25 μM to 13.1 mM, and low detection limit of 0.25 μM (S/N = 3) as a non-enzymatic ascorbic acid sensor. Whereas, for H2O2, the sensor showed a high sensitivity of 222.16 μAmM−1cm−2, wide linear range from 0.3 μM to 10.7 mM, and low detection limit of 0.3 μM (S/N = 3). The feasibility of the developed highly sensitive, selective, and reproducible H2O2 and ascorbic acid sensors was investigated in human serum. The experimental observations demonstrated that the HNONS@Tr-rGO nanocomposite is highly promising for the development of electrochemical sensors to detect biomolecules.