Chitosan microspheres have a great potential in pharmaceutical application. In this study, uniform-sized chitosan microspheres crosslinked with glutaraldehyde (CG microspheres) were prepared by Shirasu Porous Glass (SPG) membrane emulsification technique. Based on the characterizations of uniform size and autofluorescence, it was possible to develop a new detecting system for observing and quantifying the CG microspheres in rats with three different diameters (2.1, 7.2 and 12.5 microm) synchronously after oral administration. This system was a combination of scanning electron microscopy (SEM), laser scanning confocal microscope (LSCM) and flow cytometer technique, which showed the advantages of being simple, intuitionistic, repeatable and sensitive. After oral administration of three kinds of particles with different diameters, bioadhesion in gastrointestinal tract, absorption in gastrointestinal tract, distribution in systemic tissues, and biodegradation in reticuloendothelial system (RES) were studied firstly in detail. The CG microspheres showed different fates in bioadhesion, absorption and distribution according to their diameters, while the biodegradation also varied due to the different locations in RES. These original results would indicate a better way for the CG microspheres in the clinical application.