In this paper, we propose a novel location-free high-resolution optical microscopic imaging method based on micropipette-microsphere combined probe. According to optical principle, a physical model is established to determine the value of magnification. With SiO2 microspheres of different diameters such as 3.0, 4.4, 5.6, 7.5 and 10.0 μm, microscopic imaging experiments are carried out. The results show that the microstructure of un-etched DVD disk can be significantly magnified and the image contrast is obviously improved, which is consistent well with the theoretical calculation. Microscopic imaging of microstructures based on SiO2 microsphere array is realized by controlling the microspheres with a micropipette. Furthermore, a kind of micropipette-microsphere combined probe is prepared, by which location-free high-resolution optical microscopic imaging has implemented for the first time.