For sensitive and reproducible surface-enhanced Raman scattering (SERS)-based trace detection, a SERS platform with high concentration of homogeneously distributed hotspots is needed. Herein, we report a facile monolayer assembly of multispiked gold nanoparticles (MSGNPs) of various sizes over a large area. The assembly was achieved simply by tuning the concentration of MSGNPs in a suspension and subsequently drying it on a substrate under ambient conditions. A monolayer assembly for MSGNP sizes ranging from 150 to 640 nm has been demonstrated. The MSGNP assembly showed excellent sensitivity and uniformity of SERS-based trace detection, owing to the presence of high concentration of uniformly distributed interparticle and intraparticle hotspots. In particular, detection of 10 fM of a toxic dye, crystal violet, has been demonstrated. Moreover, ammonium nitrate, which is a commonly used material for homemade explosives, was detected at 1 mu M level. The MSGNP-based SERS platforms have great potential for applications in sensitive and reliable toxin and explosive detection.