In this paper, we present a detailed study of the effect of WO3 and Ni films in the presence of BP and BlueP-MDC nanomaterial layers on the sensitivity of a surface plasmon resonance (SPR) following the change in refractive index of the detection medium. Our nanostructure is based on the Kretschmann multilayer configuration composed of a BK7 prism, an Ag metallic film deposited on ZnO and a hybrid multilayer composed of WO3/Ni/2D-nanomaterials. In the first part, the study aims to optimize the effect of the thicknesses of the layers of the nanomaterials of the structure on the surface plasmon resonance. We obtain an angular sensitivity of the order of 480 deg/RIU for a figure of merit of 177.11 RIU−1 at the wavelength of 633 nm. These values are respectively 300% and 342% higher than those of a conventional SPR sensor (BK7/Ag/SM). After modeling the SPR biosensor studied in this paper, we showed how the plasmon resonance and sensitivity of the SPR sensor are affected by the refractive index of liquid biochemical elements. The proposed SPR structure can be used in highly sensitive urinary glucose detection and medical analysis applications.