Surface-enhanced Raman scattering (SERS) has been widely used to effectively detect various biological and organic molecules. This detection method needs analytes adsorbed onto a specific metal nanostructure, e.g., Ag-nanoparticles. A substrate containing such a structure (called SERS substrate) is user-friendly for people implementing the adsorption and subsequent SERS detection. Here, we report on powerful SERS substrates based on efficient fabrication of Ag-filled anodic aluminum oxide (AAO) films. The films contain many nanopores with small as-grown inter-pore gap of 15 nm. The substrates are created by electrochemically depositing silver into nanopores without an additional pore widening process, which is usually needed for conventional two-step AAO fabrication. The created substrates contain well-separated Ag-nanoparticles with quite a small inter-particle gap and a high number density (2.5 × 10¹⁰ cm⁻²). We use one-step anodization together with omitting additional pore widening to improve the throughput of substrate fabrication. Such substrates provide a low concentration detection limit of 10⁻¹¹ M and high SERS enhancement factor of 1 × 10⁶ for rhodamine 6G (R6G). The effective detection of biological and organic molecules by the substrate is demonstrated with analytes of adenine, glucose, R6G, eosin Y, and methylene blue. These results allow us to take one step further toward the successful commercialization of AAO-based SERS substrates.