The invention discloses a three-dimensional surface topography measurement method and system based on optical microscopy, which relate to the field of micro nano object measurement. The method comprises steps: a measured surface is placed in an observation range of an optical microscope, an appropriate objective lens and a lighting system are selected, an objective lens driving system is used to drive the objective lens and the measured surface to perform longitudinal relative motion, and during a serial layer scanning process, an image acquisition module is used to acquire a sequence image, and the depth position information when each image is acquired is recorded at the same time; a focusing evaluation algorithm is used to calculate the definition value of each pixel point in the sequence image; and according to the change condition of the focusing degree of the pixel point in the image sequence, a depth calculation algorithm is used to calculate the depth information of each pixel point, and thus, three-dimensional coordinates of the pixel point are thus acquired. The method and the system are applicable to the micro nano object measurement field, the surface waviness and the roughness can be measured, the three-dimensional surface topography data are rebuilt, the material surface texture information can be acquired, and the method and the system can be used for measuring a complex surface.