In the present study, the zebrafish was explored as an in vivo model to assess the biofate of transferrin receptor (TfR)-targeted coumarin 6 (C6) micelles across various biological barriers. Three 7peptide (7pep)-decorated poly(ethylene glycol)-block-poly(ϵ-caprolactone) micelles loaded with fluorescence coumarin 6 (7pep-M-C6) with different ligand densities were constructed with particle sizes between 30 and 40 nm. Whole-mount immunostaining revealed that the expression level of TfR in the retina, brain, and intestine increased along with development stage. Compared to unmodified micelles, 7pep-M-C6 demonstrated higher uptake efficiency in the larval zebrafish. Preinhibition of TfR with 7pep implicated the TfR-mediated endocytosis pathway in the uptake of 7pep-M-C6. Confocal images of the larval zebrafish eye and brain showed the efficient delivery of C6 across the retinal pigment epithelial to the ganglion cell layer and the significant accumulation of C6 in all brain tissues, respectively, which plateaued when the ligand density was 10%. To investigate the intestinal distribution of C6, micelles were administered to adult zebrafish via gavaging. Notably, 7pep-M-C6 enhanced the transport of C6 across the villi and increased its aggregation into the basolateral membrane of the intestine. After the oral administration of 7pep-M-C6, C6 accumulated in the eye and brain. Förster resonance energy transfer analysis suggested that intact 7pep-modified micelles could enter the epithelial cells of the intestine, brain, and eye after oral administration in adult zebrafish. In conclusion, zebrafish could be used as a model for in vivo visual assessment of the biofate of TfR-targeted drug delivery systems.