In the present paper, fluorescein isothiocyanate was chosen as a fluorescence probe to mark casein protein in alkaline conditions. The interaction of the casein protein marked or not marked and fluorescein isothiocyanate was preliminarily discussed by the spectrum changes of UV-absorption and fluorescence spectrometry. Fluorescent marker was separated from SephadexG-50 chromatography column. With it as an emulsifier, the fluorescently-labeled ghee microcapsules were prepared by spray drying. And using laser scanning confocal microscope by tomoscan imaging to detect the microstructure of ghee microcapsules with the excitation of 488 nm argon-ion laser, the results showed that the casein protein assembled in the membrane surface of oil-water interface and microcapsules. The ghee microcapsules had two forms, namely mononuclear and multinuclear. The microcapsule was spherical. Its surface was smooth with no crack and no hollow. Its wall surface was intact and wall structure was relatively dense. The particle size showed obvious difference. Small particles attached to large particles, forming partial agglomerating powders to contribute to enhancing the solubility of microcapsules. These prove that the ghee microcapsule is an ideal microcapsule product.