A seed-mediated electroless deposition (SMED) approach for fabrication of large-area and uniform gold nanoparticle films as efficient and reproducible as surface-enhanced Raman scattering (SERS) substrates was presented. This approach involved a seeding pretreatment procedure and a subsequent growth step. The former referred to activation of polylysine-coated glass slides in gold seed solution, and the latter required a careful control of the reactant concentration and reaction time. With the aid of gold seeds and appropriate reaction conditions, a large-area and uniform nanofilm with evenly distributed gold nanoparticles (Au NPs) was formed on the surface of the substrates after adding a mixed solution containing ascorbic acid and trisodium citrate. The morphology of the Au nanofilm was examined by scanning electron microscopy. The size evolution of Au NPs on the surface of the substrates was analyzed in detail. The nanofilm substrate was prepared by reaction conditions of the seeded activation process: 10 mL ascorbic acid and trisodium citrate mixture and 30 min of soaking time, which exhibited an excellent uniformity and reproducibility of SERS enhancement with relative standard deviation (RSD) values of less than 8% (particularly, a RSD value of 3% can be reached for the optimized measurement). Compared to the common electroless deposition, the seed-mediated electroless deposition possessed inherent advantages in controllability, reproducibility, and economic benefit.