Researchers nowadays have devoted extra attention to the different biomedical applications of indocyanine green (ICG), a US Food and Drug Administration‐approved fluorescent compound in the fields such as drug delivery, medical imaging and disease diagnosis. In addition, hepatic function evaluation could be conducted by using ICG before surgical procedures and angiographic assessment of blood. Therefore, ICG will be expected to be excellent imaging and targeting agent in various preclinical and clinical model systems. However, whether ICG possesses the potential for the gall bladder's intraoperative imaging guidance needs to be further explored in vivo animal experiments. Herein, near‐infrared fluorophores ICG can display the specific uptake by the gall bladder cells and tissues. The dynamic process of biodistribution and the clearance of ICG in vivo in mice are clearly shown in real‐time live‐body imaging. Furthermore, ICG was rapidly excreted into the bile and lately biodistributed to the stomach after treatment in mice. Meanwhile, the signal‐to‐background ratio of the gall bladder demonstrated a tremendously higher level compared to other organs (stomach, heart, liver, lung, pancreas, spleen, intestine and duodenum). In conclusion, fluorescence navigation using ICG fluorescence imaging will provide good visualization and detection of the target lesions (gall bladder) in clinics such as diagnostic medical imaging and intraoperative navigation.