AbstractOrganic nanomaterials, as nanocarrier platforms, have tremendous potential for biomedical applications. The authors successfully prepared novel two‐dimensional covalent organic nanosheets (CONs) that can be used as efficient in vivo bioimaging probes by condensing 1,3,5‐triformylglucinol (Tp) and 2,7‐diaminopyrene (Py) to produce TpPy covalent organic frameworks (COFs). TpPy COFs are then subjected to a liquid exfoliation process to obtain TpPy CONs (< 200 nm in size and < 1.7 nm in thickness). TpPy CONs disperse well in water to provide a stable, homogeneous colloidal suspension, which shows favorable photoluminescence properties. Cell viability tests using MDA‐MB‐231 and RAW 264.7 cells reveal that TpPy CONs are low in cytotoxicity. Confocal microscopy reveals clear fluorescent cell images after incubation with TpPy CONs for 24 h, without reduction in cell activity or cytosolic aggregation. To investigate the biological behavior of the TpPy CONs, the authors perform an in vivo fluorescence imaging study using MDA‐MB‐231 tumor‐bearing mice. After intravenous injection of TpPy CONs disperse in phosphate‐buffered saline (PBS), persistent and strong fluorescence signals are observed in the tumor region, with low background signals from normal tissues at 1, 3, 12, and 24 h after injection. Furthermore, these in vivo imaging results concurred with ex vivo biodistribution and histological results.