Molybdenum disulfide (MoS2) monolayer, a two dimensional (2D) layered transition metal dichalcogenides with its novel nanoelectronic and optoelectronic properties has been investigated and applied widely. MoS2-based hybrid composites have shown great potential in chemical and biological fields by combining the advantages of several structures. In our work, a SERS-active substrate was fabricated by combining the MoS2 monolayer with Ag Nanowire (NW)−Nanoparticle (NP) structures using a spin-coated method. This AgNW−AgNP−MoS2 hybrid structure was characterized by SEM, UV-Vis and Raman spectroscopy. Experimental results indicate that strong SERS signals of rhodamine 6G (R6G) molecules is able to be achieved at the “hotspot” formed in this hybrid structure. The enhancement factor is high up to 106 as the incident laser is polarized perpendicular to the NW and the limit of detection is found to be as low as 10-11 M. Besides, the fabricated SERS substrate was reliable and reproducible, which showed great potential to be an excellent SERS substrate for chemical and biological detection.