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In this paper, we study the effects of the structure, defects, and surface morphology of zinc oxide thin films on the enhancement of the Raman signal of organic molecules. The SERS substrates of Ag/ZnO thin film with low cost, stability, and high sensitivity are prepared to detect the rhodamine 6G (R6G) reagent and metronidazole (MNZ) standard. Zinc oxide (ZnO) thin films are produced by a simple and low-cost sol−gel dip-coating method. The films are dip-coated and heated at 250 °C for 45 min. The deposition process is repeated from 1 to 6 times to obtain ZnO films with various thicknesses. Following that, ZnO films are annealed at 500 °C for different duration from 30 to 150 min to obtain a good structure and porous surface which enhance the incident light scattering for SERS. The effects of different film thicknesses and annealing times on the surface morphology, structure, defects, and optical properties of ZnO thin films are investigated by field-emission scanning electron microscopy, Raman spectroscopy, ultraviolet−visible spectroscopy, and reflectance spectroscopy. The results indicate that the films with 6 layers and annealed in 120 min show the best behavior in crystallinity and surface roughness. Finally, silver nanoparticles (Ag NPs) are deposited onto the ZnO films with different sputtering times by the DC magnetron sputtering method to form the active SERS substrates. The optimal SERS sample of Ag (10 s)/ZnO (6 layers, 500 °C, 120 min) is highly sensitive, stable, and has spectral reproducibility. The SERS substrate detects R6G solution at a limit of detection (LOD) of 10 −14 M, an enhancement factor (EF) of 2.62 × 10 13 , and detects MNZ solution with a LOD of 0.01 ppm and EF of 3.30 × 10 6 . It is very stable for at least 6 months as stored away from moisture and light and has a high spectral reproducibility with the RSD of the method in three Ag/ZnO samples of 10.70, 7.34, and 10.44%.