The present invention discloses a microscopic three-dimensional measurement system and method based on a moving diaphragm. The present invention adds the diaphragm into the existing optical microscopic imaging system to limit light irradiation angle during imaging for reducing the diameter of blur circle, which extends the depth of field and the depth measurement range, so as to achieve the three-dimensional measurement of large-size objects to be measured. Through changing the position of the added diaphragm, two images with different light incident directions are obtained, which is similar to binocular stereo vision, and then the disparity map is used to predict the depth, so as to carry out the 3D scene reconstruction. Since the depth of field of the imaging system is enlarged and the imaging model has certain non-linear characteristics, the present invention uses quadratic function to express the non-linearity, which reduces the measurement error.