An effective SERS-based detection method has been developed to quantitatively diagnose the goat serum which overcomes the problem of diffusion limitation in traditional heterogeneous immunoassay. In this work, the ultra-sensitive silver/anodic aluminum oxide (Ag/AAO) SERS platform was explored via magnetron sputtering which can precisely control the sample morphology and intergap distances. Results indicated that the localized surface plasmon resonance (LSPR) effect was sharply strengthened as the sub-10 nm nanogaps generated and the enhancement factor (EF) for crystal violet (CV) was calculated to be 3.677 × 107. This novel Ag/AAO substrate with substantial “hot spots” exhibited high SERS sensitivity which could obtain extremely low limits of detection (LOD) of 10−12 M for CV. Importantly, this SERS platform was employed to detect goat serum and reached a LOD at 1 ng/μl. As a nondestructive detection technique, our SERS-based methodology required small sample quantity which expected to achieve more biomolecular detection.