Hypoxia refers to a condition where cells and tissues experience low, inadequate levels of O2. While healthy tissues are typically supplied with sufficient O2 (normoxia), cancerous tissues commonly face hypoxia due to the tumor’s extraordinarily high demand for oxygen. Various fluorescent small-molecule probes have been designed for selective detection of hypoxia in living cells, but few nanomaterials have been investigated for this type of bioimaging. Herein, we prepare a fluorescent covalent organic framework (COF) with β-ketoenamine linkages and post-synthetically modify it to conjugate hypoxia-sensitive nitroimidazole moieties into its pores (Hyp-COF). Stacks of sheets in Hyp-COF observed under electron microscopy were exfoliated by sonication, and DLS measurements confirmed particle size of less than 200 nm. Thus-prepared material exhibited good stability in physiological conditions and low cytotoxicity in in vitro experiments. Hyp-COF also showed useful fluorescence properties with an emission peak at 490 nm (λex = 420 nm) at both neutral and acidic pH levels that are characteristic of tumor tissues. Encouraged by the favorable properties of the material, we incubated HeLa cells pre-treated in either hypoxic or normoxic conditions with Hyp-COF. Fluorescence microscopy images demonstrated that the material is preferentially taken up by hypoxic cells, which show higher fluorescence signal in their interior than cells cultured under normoxia conditions. It is anticipated that this study will stimulate further developments of COFs for imaging various biological conditions.