In this research, a facile “in-situ reduction” strategy was developed to construct silver clusters-loaded silica-based hybrid nanoparticles (Ag@SHNPs). Firstly, the formation of organosilica-micellar hybrid nanostructure was achieved by self-assembly of amphiphilic block copolymer PS₈₉-b-PAA₁₆ and hydrolysis, and polycondensation of (3-mercaptopropyl)trimethoxysilane (MPTMS) on the hydrophilic PAA segment. Then, the abundant thiol groups in the organosilica framework were used as reduction sites to in-situ convert the silver salt into silver clusters, and finally the Ag@SHNPs were obtained. Morphology, structure and composition of the hybrid nanoparticles were analyzed, and their cytotoxicity on different cell lines were explored, showing good biocompatibility. The surface enhanced Raman scattering (SERS) activity of the Ag@SHNPs substrate were detected by using 4-mercaptobenzoic acid (4-MBA) as the probe molecule. Under an excitation wavelength of 532 nm laser, 4-MBA-labeled Ag@SHNPs exhibited obvious Raman enhanced signal with an enhancement factor of about 10⁵. Therefore, the silica-based hybrid substrate material shows potential application prospects in SERS bioimaging and high-sensitivity detection.