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This research introduces a novel approach using silver (Ag) nanostructures generated through electrochemical deposition and photo-reduction of Ag on fluorine-doped tin oxide glass substrates (denoted as X-Ag-Ag_yFTO, where ‘X’ and ‘y’ represent the type of light source and number of deposited cycles, respectively) for surface-enhanced Raman spectroscopy (SERS). This study used malachite green (MG) as a Raman probe to evaluate the enhancement factors (EFs) in SERS-active substrates under varied fabrication conditions. For the substrates produced via electrochemical deposition, we determined a Raman EF of 6.15 × 10⁴ for the Ag₂FTO substrate. In photo-reduction, the impact of reductant concentration, light source, and light exposure duration were examined on X-Ag nanoparticle formation to achieve superior Raman EFs. Under optimal conditions (9.0 mM sodium citrate, 460 nm blue-LED at 10 W for 90 min), the combination of blue-LED-reduced Ag (B-Ag) and an Ag₂FTO substrate (denoted as B-Ag-Ag₂FTO) exhibited the best Raman EF of 2.79 × 10⁵. This substrate enabled MG detection within a linear range of 0.1 to 1.0 µM (R² = 0.98) and a detection limit of 0.02 µM. Additionally, the spiked recoveries in aquaculture water samples were between 90.0% and 110.0%, with relative standard deviations between 3.9% and 6.3%, indicating the substrate’s potential for fungicide detection in aquaculture.