Fluorescence microscopic image three-dimensional (3-D) reconstruction is a challenging topic in image processing and computer vision, and can be widely applied to life science, biology, and medicine. A microscopic images 3-D reconstruction method is proposed for transparent or partially transparent microscopic samples, which is based on the Taylor expansion theorem and polynomial fitting. First, the image stack of the specimen is divided into several groups in an overlapping or nonoverlapping way along the optical axis, and the first image of every group is regarded as the reference image. Then, different order intensity derivatives are calculated using all the images of every group and a polynomial fitting method. Subsequently, a new image can be generated by means of Taylor expansion theorem and the calculated different order intensity derivatives and for which the distance to the reference image is Δz along the optical axis. Finally, the microscopic specimen can be reconstructed in 3-D form using deconvolution technology and all the images including both the observed and the generated images. The experimental results show the superior performance via processing simulated and real fluorescence microscopic degraded images.