DNA origami-based nanotechnology is a versatile tool for exploring fundamental biological questions and holds significant promise for future biomedical applications. Here, we leverage the optical transparency of the embryonic zebrafish to analyze live embryos injected intravenously with fluorescently labeled wireframe DNA origami nanosheets. Our approach integrated long-term, high-resolution imaging of transgenic live zebrafish embryos with single-cell RNA sequencing to elucidate the effects of oligolysine-polyethylene glycol copolymer (K-PEG) coating on the biodistribution of fluorescence signal in embryos injected with wireframe DNA origami nanosheets. We observed rapid accumulation of fluorescence signal in the caudal hematopoietic tissue (CHT). K-PEG coating mitigated the accumulation of fluorescence signal in CHT, enabling increased detection of signal in other tissues. Our findings highlighted the pivotal role of scavenger endothelial cells in DNA origami clearance, with K-PEG enabling the prolonged detection of fluorescence signal at the CHT. Furthermore, using a transgenic zebrafish line designed for targeted macrophage ablation, we found that macrophages contribute to the clearance of fluorescence signal in embryos injected with the noncoated but not with K-PEG-coated nanosheets. This study introduces a framework for the analyses of the biodistribution and clearance of DNA origami nanostructures in vivo with single-cell resolution in zebrafish models.