block (polyethylene glycol (PEG)) and hydrophobic block (such as polycaprolactone (PCL), poly lactic-co-glycolic acid, and poly-d,l-lactic acid) have promising applications to deliver water-insoluble drugs owing to their stealth shell-hydrophobic core structure. However, to date, only a few polymeric NCs (excluding biologics) were approved in clinics, such as GenexolPM/Cynviloq (Samyang Biopharm), Nanoxel (Fresenius Kabi), and Apealea/Paclical (Oasmia Pharmaceuticals). [2] There exists a large gap from basic research to the clinical transformation of polymeric NCs because they cannot offer a significantly improved therapeutic benefit over the free drug. [3][4][5] Once entering the blood circulation, polymeric NCs will face a complex biological environment (e.g., dilution, shear force, and protein adsorption) and various plasma components, which will dramatically affect their biofate. [4] Polymeric NCs may lose their integrity in blood circulation. Intact polymeric NCs may be recognized and rapidly sequestered by reticuloendothelial system (RES) and further eliminated from the systemic circulation. However, due to the lack of sufficient understanding of their biofate and parameters that could regulate in vivo processes, the behaviors of polymeric NCs in vivo could not be well controlled, resulting in unsatisfactory therapeutic benefit.Moreover, the in vivo characterization and optimization of NCs are also challenging in early formulation development in the absence of a proper screening model. The in vivo fate of polymeric NCs not only depends on the biological environment but also relies on their physicochemical properties, such as particle size, shape, surface modification, and charge, which offer enormous possibilities for formulation optimization. [3,6,7] Although in vitro cell models are conventional drug screening tools, they cannot completely reflect or mimic the in vivo biological environment, resulting in formulations screened in cell models that might not work in rodent models. [8] Moreover, rodent experiments are time-consuming and costly, failing to assess a large number of formulations in a short time. Thus, there exists an urgent demand for a suitable preclinical model Polymeric nanocarriers have high biocompatibility for potential drug delivery applications. After entering bloodstream, nanocarriers will circulate, interact with proteins, dissociate, or be cleared by reticuloendothelial system. Zebrafish as a visual animal model, can serve as a tool for screening nanomedicines and monitoring nanocarrier behaviors in vivo. However, a comprehensive correlation between zebrafish and rodent models is currently deficient. Here, different-sized poly(caprolactone) nanocarriers (PCL NCs) are fabricated with or without PEGylation to investigate correlation between zebrafish and mice regarding their biofate via Förster resonance energy transfer technique. Results show that PEGylated PCL NCs have higher integrity in both zebrafish and mice. Small PEG-PCL NCs have longer circulation, while large PEG-PCL NCs have ...