Mo-48.2% Ag films were fabricated by direct current (DC) magnetron sputtering and annealed in an argon atmosphere. The effects of annealing on the surface morphology, resistivity and surface-enhanced Raman scattering (SERS) performance of Mo-48.2% Ag films were investigated. Results show a mass of polyhedral Ag particles grown on the annealed Mo-48.2% Ag films’ surface, which are different from that of as-deposited Mo-Ag film. Moreover, the thickness and the resistivity of Mo-48.2% Ag films gradually decrease as the annealing temperature increases. Furthermore, finite-difference time-domain (FDTD) simulations proved that the re-deposition Ag layer increases the “hot spots” between adjacent Ag nanoparticles, thereby greatly enhancing the local electromagnetic (EM) field. The Ag layer/annealed Mo-48.2% Ag films can identify crystal violet (CV) with concentration lower than 5 × 10⁻¹⁰ M (1 mol/L = 1 M), which indicated that this novel type of particles/films can be applied as ultrasensitive SERS substrates.