Characterizing the transient ultratrace light-independent intracellular singlet oxygen (1O2), which plays a vital role in multiple biological processes in living organisms, brings about tremendous help for understanding the nature of 1O2-mediated or related bioevents. Nevertheless, an approach to detect the light-independent intracellular 1O2 is hard to find. Herein, we developed a chemiluminescent nanosensor by compacting a great number of TPE-N(Ph)-DBT-PH molecules in one nanostructure via autoaggregation. Taking advantage of the aggregation-induced emission property, this TPE-N(Ph)-DBT-PH nanosensor is highly fluorescent and promises a bright red-light CL and the convenience of mapping in vivo sensor distribution. Experiments demonstrate the nanosensor’s unprecedented selectivity toward 1O2 against other reactive oxygen species. The 3.7 nmol L–1 limit of detection renders this nanosensor with the best-known sensitivity of 1O2 chemical sensors. Meanwhile, fluorescence confocal microscope imaging results suggest that our nanosensor simultaneously targets mitochondria and lysosomes in RAW 264.7 cells via the energy-dependent endocytosis pathway, thereby implying an attractive potential for the detection of intracellular 1O2. Such a potential is demonstrated by detecting 1O2 in RAW 264.7 cells during a lipopolysaccharide and phorbol myristate acetate stimulated respiration burst. This study represents the first approach to detect light-independent intracellular 1O2 during cell bioregulation. Thus, our nanosensor provides an effective tool for investigating the 1O2-related bioprocesses and pathological processes.