Fluorescent microspheres are widely used in the field of biomedicine. This article presents a novel and facile microfluidic method to prepare monodispersed chitosan microspheres with relatively small diameter and enhanced autofluorescence properties. Highly monodispersed chitosan microspheres crosslinked with glutaraldehyde (GS), formaldehyde (FS) and their mixture (MS) with average diameter from 20 mu m to 8 mu m were prepared by controlling the flow rate of the continuous phase via a co-axial capillary microfluidic device. The fluorescence intensity of MS prepared microspheres was enhanced more than that of the GS/FS prepared microspheres because of the aldol reaction between formaldehyde and glutaraldehyde. The optimum ratio of formaldehyde to glutaraldehyde was only 1:49 resulting in a fluorescence intensity enhancement as high as 160%. The tunability of the fluorescent intensity and particle size makes the microspheres versatile probes and tracers for a variety of different biomedical applications. (C) 2012 Elsevier B.V. All rights reserved.