By combining the excellent biocompatibility of molybdenum disulfide (MoS2), excellent surface-enhanced Raman scattering (SERS) activity of Au nanoparticles (Au NPs), and large surface area of Si nanosquare holes (NSHs), a structure in which MoS2 is decorated with Au NPs on Si NSHs, was proposed for SERS applications. The NSH structure fabricated by e-beam lithography possessed 500 nm of squares and a depth of approximately 90 nm. Consequently, a few-layer MoS2 thin films (2–4 layers) were grown by the sulfurization of the MoO3 thin film deposited on Si NSHs. SERS measurements indicated that MoS2 decorated with Au NPs/Si NSHs provided an extremely low limit of detection (ca. 10−11 M) for R6G, with a high enhancement factor (4.54 × 109) relative to normal Raman spectroscopy. Our results revealed that a large surface area of the NSH structure would probably absorb more R6G molecules and generate more excitons through charge transfer, further leading to the improvement of the chemical mechanism (CM) effect between MoS2 and R6G. Meanwhile, the electromagnetic mechanism (EM) produced by Au NPs effectively enhances SERS signals. The mechanism of the SERS enhancement in the structure is described and discussed in detail. By combining the hybrid effects of both CM and EM to obtain a highly efficient SERS performance, MoS2 decorated with Au NPs/Si NSHs is expected to become a new type of SERS substrate for biomedical detection.