Three-dimensional (3D) reconstruction is extensively used in microscopic applications. Reducing excessive error points and achieving accurate matching of weak texture regions have been the classical challenges for 3D microscopic vision. A Multi-ST algorithm was proposed to improve matching accuracy. The process is performed in two main stages: scaled microscopic images and regularized cost aggregation. First, microscopic image pairs with different scales were extracted according to the Gaussian pyramid criterion. Second, a novel cost aggregation approach based on the regularized multi-scale model was implemented into all scales to obtain the final cost. To evaluate the performances of the proposed Multi-ST algorithm and compare different algorithms, seven groups of images from the Middlebury dataset and four groups of experimental images obtained by a binocular microscopic system were analyzed. Disparity maps and reconstruction maps generated by the proposed approach contained more information and fewer outliers or artifacts. Furthermore, 3D reconstruction of the plug gauges using the Multi-ST algorithm showed that the error was less than 0.025 mm.