Adenosine triphosphate (ATP) is an important biomolecule, which is the primary source of cellular energy. In particular, an abnormal metabolism of ATP level has been took part in many diseases, such as cancer. Thus, developing an effective fluorescent probe for tumor-targeting imaging of ATP is great importance for in-depth understanding the functions of ATP in tumor invasion and matastasis. In this work, we present the design and synthesis of a tumor-targeting near-infrared (NIR) fluorescent probe named Bio-SiR. Bio-SiR is mainly composed of three parts: si-rhodamine-based fluorophore, diethylenetriamine-based recognition group and biotin-based tumor-targetable group. When Bio-SiR reacts with ATP, a turn-on fluorescence at 675 nm (NIR region) is observed clearly, which is suitable for its application in mice. In addition, due to a concurrent effect from dual recognition sites, the probe Bio-SiR displays excellent selectivity for ATP over other potential biological analytes. Under the guidance of biotin group, Bio-SiR can be successfully used for imaging ATP in cancer cells. Furthermore, live-cell imaging allows us to directly real-time monitor the dynamic change of ATP in cancer cells. In particular, this is the first tumor-targeting NIR small-molecule fluorescent probe for endogenous ATP imaging in tumor-bearing mice. These features demonstrate that this probe is a useful imaging tool for expounding the function of ATP in cancer.