Edge enhancement and extraction of biological samples′ images are one of the key technologies for the processing of medical images, and the basis of samples′ morphological analysis. In general, the realization for image edge enhancement is through post processing of original images by computer programming. However, spatial differential confocal microscopy system has been applied to directly obtain the high resolution and contrast edge enhanced microscopic images of samples while the confocal microscopic images are obtained. The biomedical application is demonstrated not only for edge enhancement imaging for a mask and a resolution test target (RTA-07) that shows the resolution of the system is 1.5 μm, but also for edge enhancement imaging for biological cells like RBCs and oral epithelial cells. The obtained edge enhanced microscopic images can be used for simply sample morphological analysis and calculation, such as the scale and area, which has a practical application significance in the research of biomedicine.