The line defects of two-dimensional (2D) transition metal dichalcogenides (TMDs) play a vital role in determining their device performance. In this work, a microscopic hyperspectral imaging technique based on differential reflectance was introduced for the online inspection of line defects in TMDs. Upon comparison of the measurement results of imaging and spectra, the relationship between optical contrast and differential reflectance spectra was established. A light selection method was proposed to optimize the optical contrast of line defects. Via application of an image processing algorithm, an automatic detection of the line defects with a classification accuracy of 95% was achieved for WS2, MoS2, and MoSe2. This work not only provides a microscopic hyperspectral imaging technique for detecting 2D material defects but also introduces a versatile design strategy for developing an advanced machine vision spectroscopic system.