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The development of SERS detection technology is challenged by the difficulty in obtaining SERS active substrates that are easily prepared, highly sensitive, and reliable. Many high-quality hotspot structures exist in aligned Ag nanowires (NWs) arrays. This study used a simple self-assembly method with a liquid surface to prepare a highly aligned AgNW array film to form a sensitive and reliable SERS substrate. To estimate the signal reproducibility of the AgNW substrate, the RSD of SERS intensity of 1.0 × 10⁻¹⁰ M Rhodamine 6G (R6G) in an aqueous solution at 1364 cm⁻¹ was calculated to be as low as 4.7%. The detection ability of the AgNW substrate was close to the single molecule level, and even the R6G signal of 1.0 × 10⁻¹⁶ M R6G could be detected with a resonance enhancement factor (EF) as high as 6.12 × 10¹¹ under 532 nm laser excitation. The EF without the resonance effect was 2.35 × 10⁶ using 633 nm laser excitation. FDTD simulations have confirmed that the uniform distribution of hot spots inside the aligned AgNW substrate amplifies the SERS signal.