The applications of nanotechnology for drug delivery systems(DDSs) have increased rapidly. To facilitate this rapid development, an ideal model that is suitable for high-throughput screening(HTS) and transport mechanism elucidation of the novel formulation across biological barriers including blood brain barrier(BBB), gastrointestinal(GI) barrier, blood retinal barrier(BRB), as well as tracking the in vivo biofateis desperately needed. In vitro cell culture models have been widely used, although they are far from a complimentary in vivo system. Mammalian animal models are common predictive models, but they are costly and time consuming. Zebrafish(Danio rerio), a small vertebrate model, which have the transparency body in the early development stages and similar biological barriers in structure and function, have the potential to be developed as an in vivo model for quick and visual evaluation of DDSs. However, its application has not been fully explored in pharmaceutics, especially the development of DDSs. In this study, we firstly investigated the transport of two different sized coumarin 6 nanocrystals(C6-NCs) across biological barriers at several developmental stages of zebrafish. Higher uptake and transport efficiency was observed with small sized C6-NCs. Along with the maturation of these biological barriers, 7 days postfertilization(dpf) zebrafish showed its unique advantages to be developed for the visible evaluation of transport of DDSs across biological barriers. Particle size also affects the in vivo behavior of C6-NCs, especially in the brain, where lipid rafts participated in the transport of small sized NCs in adult zebrafish. These results demonstrated the successful application of zebrafish as a simple and dynamic model to assess the transport and biodistribution of DDSs and showed its potential to be used for central nervous system(CNS) DDSs screening. Next, we investigated the biofate of transferrin receptor(TfR)-targeted 7 peptide(7 pep)-decorated C6 micelles(7 pep-M-C6) with different ligand density with the aim to develop zebrafish as an in vivo model for the visual evaluation of the biofate of TfR targeted DDSs. TfR expression pattern is varied with the developmental stages and tissues. Intestine, eye and brain are the main sites of TfR. 7 pep modification enhanced the uptake of micelles in larval zebrafish through endocytosis in a ligand densitydependent manner with saturable process. The delivery of C6 across the biological barriers was also improved after 7 pep modification. F?rster Resonance Energy Transfer(FRET) analysis combined with the lambda scan revealed that during the absorption of 7 pep-M-C6 in the intestine after oral administration, and some intact micelles could transport across the GI barrier and reach the posterior segment of eye and brain. In conclusion, more information about the in vivo behavior of these two DDSs was provided in this model and strong evidence supported that zebrafish has potential to be developed as an in vivo model for quick evaluations of transport and in vivo biofate of DDSs.