Subsurface damage (SSD) detection and removal play an important role in improving the laser induced damage threshold (LIDT) of optics. A new method to detect the distribution of SSD is proposed. This method combines total internal reflection microscope and digital image processing technology. Because of the limited focal depth of the microscope system, one can get different images focusing at different depth levels by a total internal reflection microscopy (TIRM). The connection between the definition curve of TIRM image sequence and the depth position of SSD is established. Also, the depth length of SSD through the feature of definition curve is obtained. The imaging process of TIRM is simulated and the typical features of the focusing process is found out. Size information of the microstructures is got exactly according to the fabricated microstructures by femtosecond laser micromachining. Focal depth of the microscope determines the test depth accuracy of our method and actually the test accuracy can achieve 1 μm.