This study utilized semiconductor processing techniques to fabricate patterned silicon (Si) substrates with arrays of inverted pyramid-shaped micro-pits by etching. Molybdenum trioxide (MoO3) was then deposited on these patterned Si substrates using a thermal evaporation system, followed by two-stage sulfurization in a high-temperature furnace to grow MoS2 thin films consisting of only a few atomic layers. During the dropwise titration of Rhodamine 6G (R6G) solution, a longitudinal electric field was applied using a Keithley 2400 (Cleveland, OH, USA) source meter. Raman mapping revealed that under a 100 mV condition, the analyte R6G molecules were effectively confined within the pits. Due to its two-dimensional structure, MoS2 provides a high surface area and supports a surface-enhanced Raman scattering (SERS) charge transfer mechanism. The SERS results demonstrated that the intensity in the pits of the few-layer MoS2/patterned Si SERS substrate was approximately 274 times greater compared to planar Si, with a limit of detection reaching 10−5 M. The experimental results confirm that this method effectively resolves the issue of random distribution of analyte molecules during droplet evaporation, thereby enhancing detection sensitivity and stability.