In this work, three-dimensional ordered macroporous (3DOM) TiO₂ was designed by assembling Ag nano-particles (NPs) into the network of 3DOM TiO₂ to form a 3DOM composite nano-architecture, the 3DOM-TiO₂@Ag. The designed 3DOM-TiO₂@Ag nano-architecture effectively amplified the Raman signal of Eosin Y (EY), owing to its interconnected 3DOM network, appropriate energy matching between EY and the 3DOM nano-architecture, and the surface plasmon resonances and optical antenna effect of Ag NPs. Firstly, interconnected 3DOM network improved the adsorption ability of 3DOM-TiO₂@Ag for EY molecules as its interconnected 3DOM networks offered high surface area and porous volume which can drive EY solution into it by large capillarity forces. Secondly, energy matching between EY molecules and each components of 3DOM-TiO₂@Ag promoted the photo-generated electrons to transfer from excited EY molecules through the Ag/TiO₂ heterojunction and inject into the Ag NPs to improve its surface electron density. Surface plasmon resonances of the Ag NPs also contributed to amplifying the Raman signals of the EY molecules. Detection limit of the 3DOM-TiO₂@Ag substrate could 10^− 12 M for EY molecules. In addition, the designed 3DOM substrate exhibited favorable stability, reproducibility and universality for detecting EY and other with structural similar molecules.