The aim of the present study was to evaluate cellular uptake of oleoyl-chitosan (OCH) nanoparticles by using A549 cells, a human lung carcinoma cell line, for drug and gene delivery applications. In this study, self-assembled OCH nanoparticles encapsulating a fluorescent marker molecule, fluorescein isothiocyanate (FITC), were prepared and characterized. The effects of particle size, concentration, and incubation time on the cellular uptake of the nanoparticles (FITC-OCH nanoparticles) were quantified by spectrofluorometric measurement and confirmed using fluorescence microscopy studies. The nanoparticles were taken up by the cells, and levels of binding and uptake increased with the decrease of particle size and the increase of particle concentration and incubation time. These results implied that the OCH nanoparticles have great potential to be applied as a drug carrier system to deliver drugs into the cells.