Our previous study prepared loading 10,11-methylenedioxy camptothecin (MD-CPT) hyaluronic acid nanocarriers (HANs) as a transdermal delivery system. The further study targets to value the cellular uptake and pharmacokinetic analysis of MD-CPT-loaded HA nanoemulsion as transdermal delivery carriers. A sustained-release dosage formulation was obtained with good skin permeability by optimizing the preparation conditions. The cellular-uptake of keloid fibroblast for nanoemulsion was observed with CLSM, drug eventually into the nucleus. Phagocytosis was time dependence, and different cell lines include HSF, HUVES, MCF-7 and KF were slightly different. Rhodanmine B labeled HANs performed desirable skin permeable capacity across stratum corneum, and the drug was transferred to the dermis by fluorescence microscopy. The optimized HANs formulation was verified with the highest effective drug permeability. The plasma concentration of MD-CPT was analyzed by HPLC, showed an almost 3.6 and 1.6 times increase T_1/2 respectively compared with intravenous and intramuscular injection; moreover, the curve of transdermal delivery was smooth continuous. In vivo imaging system intuitively reflected the distribution of transdermal drug in mice and the drug content of various organs/tissues. The rest of MD-CPT and HANs entered into the blood circulation, eventually excreted through metabolism without body burden. The longer drug residence at topical area with treatment could provide a continuous and controllable, extended efficacy, which was beneficial to superficial lesions therapy.