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Based on fuzzy measurement of microscopic optical images, analysis of the focused and defocused characteristics of the images was performed, and a method based on Depth from Defocus (DFD) is developed for micro scale image analysis and reconstruction. In this method, we use gradient operators as the measurement operator. With the establishment of relationship between the defocus measurement and the depth transformation of the micro image sequence, we obtained the depth information of the target-object. As a test of validity of our method, we also performed an experiment to measure the parallelism of a micro manipulation platform. In additional, we also reconstructed micro contour images using the DFD method and the experiment results showed that the method is valid for 3D reconstruction of microscope images.