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The authors report, for the first time, the investigation of loading and release of guest molecules from the hollow mesoporous silica (HMS) spheres by using confocal laser scanning microscopy (CLSM) technique. The HMS spheres with particle size of 800-1200-nm and peak pore size of 2.5-nm were well prepared by using the carbon spheres templating method. Using rhodamine 6G (Rho-6G) dye as guest molecules, the Rho-6G distribution in the HMS spheres after loading and time-dependent release from the HMS spheres were evaluated by the CLSM imaging and fluorescence intensity analysis. The results indicated that the guest molecules can diffuse into the hollow cores through the mesoporous channels on the shells, and the release of guest molecules from HMS spheres follows a diffusion-driven behaviour and exhibits a sustained release manner. Therefore this kind of HMS spheres as carriers has the potential for drug delivery.