A method and an apparatus for calibrating an image sensor array in a microscopic imaging system are provided. The method includes: performing a vignetting effect calibration and correction on the image sensor array, such that pixels acquired by all sub-field-of-view image sensors for capturing a scene with a same radiant exitance have a same gray level; performing a light encoding on a temporal-spatial union structure using a spatial light modulator, to establish correspondences of a plurality of feature points between an image space and a physical space; performing a light decoding on the temporal-spatial union structure, to acquire pixel coordinates of the plurality of feature points in an image plane and physical coordinates of the plurality of feature points in a plane of the spatial light modulator; acquiring a homography relationship according to the pixel coordinates and the physical coordinates, acquiring a global coordinate mapping according to the homography relationship.