This article introduced a filter paper-based SERS substrate through soaking method, and investigated the relationship between the distributions of silver nanoparticles (AgNPs) on the filter paper with soaking time in detail. SERS spectra of seminal plasma under different excitation wavelengths (514 nm and 785 nm) were compared, and the latter was confirmed to demonstrate better SERS signals. SERS and normal Raman spectra of seminal plasma using 785 nm excitation laser was compared as well. More importantly, the intensity of the strongest peak (654 cm-1) from SERS spectra of seminal plasma was used to evaluated the enhancement effects and signal reproducibility with filter paper SERS substrates under different soaking time(6 h,12 h,24 h). Our results indicated that the AgNPs on the paper substrate soaked with concentrated AgNP colloid presented a relatively uniform distribution at the soaking time of 12 h and the substrate demonstrated high Raman signal enhancement and good signal reproducibility as well.