Mechanical engineering of 2D materials allows continuous and reversible modulation of their electronic and photonic properties. Although photoluminescence (PL) measurement is an effective way to monitor the effects of mechanical forces on 2D semiconductors, there is currently a lack of techniques to enhance PL signals during stress application. This study presents an innovative mechanical engineering approach that integrates a dielectric microsphere as an atomic force microscopy (AFM) probe into a Raman-AFM system. Force–distance curve tests and COMSOL simulations were performed to analyze and estimate the compressive stress exerted by the microsphere. Importantly, the PL signals of transition metal dichalcogenides subjected to microsphere probe's force were enhanced and reveal distinct mechanical responses depending on the substrate rigidity: compressive pressures for rigid substrates and tensile strains for flexible ones. Notably, this strategy not only amplifies spectral signals in real time but also achieves fine stress modulation in the precise targeted material region, demonstrating its superiority in sensitive mechanical engineering applications. Our work offers a new avenue for the deliberate design of mechanical strains in 2D materials, which is crucial for optimizing the performance of related devices.