The laser dye 6-coumarin was selected as model of low water soluble drug to be encapsulated in glycol chitosan nanoparticles intended for transmucosal applications and, at the same time, being a fluorescent probe, it is of aid to elucidate the intracellular fate of the particles. To increase the aqueous solubility of the tracer, the complexation with different cyclodextrins was adopted. The fluorescence properties of the inclusion complexes were evaluated. The increase in aqueous solubility provided by different cyclodextrins [up to 1.4 × 10−4 M in the case of heptakis (2,6-di-O-methyl)-β-cyclodextrin] allowed the preparation of novel glycol chitosan nanoparticles according to the ionic cross-linking of the polycation by sodium tripolyphosphate. Small changes in the preparation technique allowed to produce particles of two different sizes, around 200 nm and bigger than 300 nm where the contribution of cyclodextrin consisted of the modulation encapsulation efficiency in the final particles. Confocal laser scanning microphotographs clearly showed the internalization of 6-coumarin nanoparticles in Caco-2 cell line. The results reveal that these biodegradable nanoparticles hold promise as probes in biomedical field.