A homogeneous monolayer film of the silver nanoparticles was prepared, and a highly active surface-enhanced Raman scattering substrate was obtained. Using rhodamine 6G (R6G) as a probe molecule, the SERS characteristic of the silver nanoparticles monolayer film was compared with both SERS substrates by direct dropping silver colloid mixing with and without R6G, respectively. It was found that the monolayer film exhibited a high sensitivity (The detection limit of R6G was 10-9 mol/L) and reproducibility. The enhanced reason was analyzed by finite difference time domain (FDTD) method. The results showed that the experimental results and theoretical simulation were in line.