J-aggregation is an efficient strategy for the development of fluorescent imaging agents in the second near-infrared window. However, the design of the second near-infrared fluorescent J-aggregates is challenging due to the lack of suitable J-aggregation dyes. Herein, we report meso-[2.2]paracyclophanyl-3,5-bis-N,N-dimethylaminostyrl BODIPY (PCP-BDP2) as an example of BODIPY dye with J-aggregation induced the second near-infrared fluorescence. PCP-BDP2 shows an emission maximum at 1010 nm in the J-aggregation state. Mechanism studies reveal that the steric and conjugation effect of the PCP group on the BODIPY play key roles in the J-aggregation behavior and photophysical properties tuning. Notably, PCP-BDP2 J-aggregates can be utilized for lymph node imaging and fluorescence-guided surgery in the nude mouse, which demonstrates their potential clinical application. This study demonstrates BODIPY dye as an alternate J-aggregation platform for developing the second near-infrared imaging agents.

Detection and imaging of α‐L‐fucosidase (AFU) is of great value to understand its roles in hepatocellular carcinoma (HCC) and tumor early diagnosis, but ideal assays are still lacking. Herein, a near‐infrared (NIR) fluorescent biosensor (α‐Fuc‐DCM) was elaborately designed and synthesized for rapid and ratiometric detection of AFU activity in cells and HCC tumor mouse models. In the presence of AFU, this biosensor shows an enhancement in NIR emission in a ratiometric manner, which significantly improves the detection accuracy with the limit of detection as low as 4.8 mU/mL. Taking advantage of these merits, the activity of AFU in lysosomes could be visualized using ratiometric and NIR dual modality in living cells. Furthermore, its remarkable application for monitoring of endogenous AFU activity in HCC tumor‐bearing mouse model is also demonstrated with bright fluorescence signal, which indicated that the biosensor could clearly monitor the liver tumor in the early stage. Importantly, the α‐Fuc‐DCM probe can be utilized to detect the AFU in serum from HCC patients. This strategy offers a promising biosensor system for early diagnosis of HCC and studying the roles of AFU in cancers.