The availability of sensitive, reproducible, and stable substrates is critically important for surface-enhanced Raman spectroscopy (SERS)-based applications, but it presently remains a challenge. In this work, well-aligned zinc gallate (ZnGa2O4) nanorod arrays grown on a Si substrate by chemical vapor deposition were used as templates to fabricate SERS substrates by deposition of Ag nanoparticles onto the ZnGa2O4 nanorod surfaces. The coverage of the Ag nanoparticles on the ZnGa2O4 nanorod surfaces was easily controlled by varying the amount of AgNO3. SERS measurements showed that the number density of Ag nanoparticles on the ZnGa2O4 nanorod surfaces had a great effect on SERS activity. The SERS signals collected by point-to-point and SERS mapping images showed that as-prepared SERS substrates exhibited good spatial uniformity and reproducibility. Detection of melamine molecules at low concentrations (1.0×10−7 M) was used as an example to show the possible application of such a substrate. In addition, the effect of benzoic acid on the detection of melamine was also investigated. It was found that the SERS signal intensity of melamine decreased greatly as the concentration of benzoic acid was increased.