Abstract Band structure engineering by local strain of 2D transition metal dichalcogenides (TMDCs) is proven as an efficient means for improving the material performance in nanoscale light sources, photodetectors and flexible electronic devices. However, photoluminescence‐based techniques continue encountering challenges in detecting changes in band structures, mainly because of the indirect bandgap nature of the multilayer TMDCs as well as the exciton diffusion at room temperature. Herein, it is demonstrated that with the help of plasmonic nanostructures, dark‐field‐scattering‐based single‐particle plasmon spectroscopy can reveal the Fano interference between well aligned localized surface plasmon resonances and the 2D semiconductor excitonic or interband‐transition absorptions, therefore enabling precise detection of local band structure modulation in few‐layer and multilayer TMDCs under mechanical stress. By measuring the scattering spectra of individual plasmonic nanostructures covered by WS 2 , it is shown that the local strain results in an up‐to‐50 meV shift for the direct K–K transition. The accuracy of the method is further confirmed by plasmon‐enhanced photoluminescence examinations. It is believed that the results offer a robust and high‐precision method to probe the local band structure change in 2D semiconductors, which will greatly boost the development of novel 2D optoelectronic devices.