Thermally activated delayed fluorescence (TADF) materials have provided new strategies for time‐resolved luminescence imaging (TRLI); however, the development of hydrophilic TADF luminophores for specific imaging in cells remains a substantial challenge. In this study, a mitochondria‐induced aggregation strategy for TRLI is proposed with the design and utilization of the hydrophilic TADF luminophore ((10‐(1,3‐dioxo‐2‐phenyl‐2,3‐dihydro‐1H‐benzo[de]isoquinolin‐6‐yl)‐9,9‐dimethyl‐9,10‐dihydroacridin‐2‐yl)methyl)triphenylphosphonium bromide
(NID‐TPP)
. Using a nonconjugated linker to introduce a triphenylphosphonium (TPP
+
) group into the 6‐(9,9‐dimethylacridin‐10(9
H
)‐yl)‐2‐phenyl‐1H‐benzo[
de
]isoquinoline‐1,3(2
H
)‐dione
(NID)
TADF luminophore preserves the TADF emission of
NID‐TPP
.
NID‐TPP
shows clear aggregation‐induced delayed fluorescence enhancement behavior, which provides a practical strategy for long‐lived delayed fluorescence emission in an oxygen‐containing environment. Finally, the designed mitochondrion‐targeting TPP
+
group in
NID‐TPP
induces the adequate accumulation of
NID‐TPP
and results in the first reported TADF‐based time‐resolved luminescence imaging and two‐photon imaging of mitochondria in living cells.
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