Sensitive substrate is the key factor, which may affect the enhancement effect of the SERS detection. SiO2/Ag hybrid nanospheres were synthesized by using (3-Aminopropyl) trimethoxysilane (APTES) modified SiO2 nanospheres and [Ag(NH3)(2)](+) ions, which would be used as very sensitive and homogeneous surface enhanced Raman scattering (SERS) substrates. Our results show that the -NH2 groups were grafted on the surface of SiO2 due to the modification of APTES, which promoted adsorption of Ag nanoparticles on the SiO2 matrix. The effects of APTES on the micromorphology, chemical structure, and SERS performance of SiO2/Ag hybrid nanospheres were investigated by the SEM, XRD, and SERS measurements. Results indicated that the Ag nanoparticles were crystalline and the diameter of AgNPs ranged from 50 to 100 nm. SERS measurement showed that the surface electromagnetic enhancement would increase with number of AgNPs on SiO2 nanospheres surface, and the mechanism explained by the FDTD simulation. Furthermore, our results indicate the highly uniformity and reproducibility of these substrates with the relative standard deviation (RSD) of peak intensity was 7.74%. The SiO2/Ag nanospheres with had high sensitivity, uniformity and reproducibility should be potential SERS applications.