The fluorophores with long‐lived fluorescent emission are highly desirable for time‐resolved fluorescence imaging (TRFI) in monitoring target fluorescence. By embedding the aggregates of a thermally activated delayed fluorescence (TADF) dye, 2,3,5,6‐tetracarbazole‐4‐cyano‐pyridine (CPy), in distearoyl‐sn‐glycero‐3‐phosphoethanolamine‐poly(ethylene glycol) (DSPE‐PEG2000) matrix, CPy‐based organic dots (CPy‐Odots) with a long fluorescence lifetime of 9.3 μs (in water at ambient condition) and high brightness (with an absolute fluorescence quantum efficiency of 38.3%) are fabricated. CPy‐Odots are employed in time‐resolved and confocal fluorescence imaging in living Hela cells and in vivo. The green emission from the CPy‐Odots is readily differentiated from the cellular autofluorescence background because of their stronger emission intensities and longer lifetimes. Unlike other widely studied DSPE‐PEG2000 encapsulated Odots which are always distributed in cytoplasm, CPy‐Odots are located mainly in plasma membrane. In addition, the application of CPy‐Odots as a bright microangiography agent for TRFI in zebrafish is also demonstrated. Much broader application of CPy‐Odots is also prospected after further surface functionalization. Given its simplicity, high fluorescence intensity, and wide availability of TADF materials, the method can be extended to develop more excellent TADF Odots for accomplishing the challenges in future bioimaging applications.