Acetylated-chondroitin sulfate/chlorin e6 conjugates (Ac-CS/Ce6 1, 2, 3) were synthesized via the formation of an ester linkage between CS and Ce6 and evaluated as nanoscale drugs for photo dynamic therapy. Ac-CS/Ce6 2 and 3 with higher Ce6 contents of 11.7 and 17.6%, respectively, had average diameters of < 150 nm and were very stable in phosphate-buffered saline (PBS) for 1 month. The critical self-quenching concentration (CQC) of Ac-CS/Ce6 decreased as the conjugated-amount of Ce6 increased. All samples displayed autophotoquenching properties in aqueous solution, whereas their fluorescence intensity strongly correlated with the amount of Ce6 in the organic solvent dimethyl sulfoxide (DMSO). Compared with free Ce6, Ac-CS/Ce6 nanodrug photoactivity was maintained in terms of fluorescence properties and singlet oxygen (O-1(2)) generation. In a He La cell culture system, we observed rapid cellular uptake of the Ac-CS/Ce6 nanodrug without any other ligands using confocal imaging and fluorescence-activated cell sorting (FACS) analysis. Upon light irradiation following cellular uptake, phototoxicity was detected via 3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide (MTT) assay. The self-quenching effect and fluorescence recovery of Ac-CS/Ce6 were also determined both in vitro and in vivo, Taken together, our results indicate that Ac-CS/Ce6 has potential as an effective photo dynamic therapy (PDT) prodrug for clinical application.