Quantification of 25-hydroxy vitamin-D3 (25-OHVD3) is important because its deficiency is related to numerous diseases, including osteoporosis, depression, diabetes, heart disease, certain autoimmune conditions, and even Covid-19. In the present study, noble metal-metal oxide nanohybrid based on L-cysteine functionalized gold decorated zirconia nanoparticles (Cys-Au@ZrO2 NPs) were synthesized with the objective of enhanced electrochemical behavior, stability, availability of functional group to covalently bind with biomolecules, and developing an efficient immunosensor for 25-OHVD3 detection. The formation of Au@ZrO2 NPs and further Cys functionalization was validated by various characterizations. Cys-Au@ZrO2 NPs were electrodeposited onto ITO substrate and further modified with antibodies specific to 25-OHVD3 (ab-25VD3) and bovine serum albumin (BSA) to build an immunosensing platform for 25-OHVD3 detection. The fabricated BSA/ab-25VD3/Cys-Au@ZrO2/ITO immunosensor demonstrated an improved sensitivity of 2.01 μA ng−1 ml cm−2, LOD of 3.54 ng ml−1 for the linear detection range of 1–50 ng ml−1 with regression constant of 0.98. Moreover, the analytical performance of the BSA/ab-25VD3/Cys-Au@ZrO2/ITO immunosensor in determining 25-OHVD3 in human serum samples collected from four healthy people yielded satisfactory results that were highly correlated with the conventional enzyme-linked immunosorbent assay (ELISA).