Imaging cancer specific biomarkers with near-infrared (NIR) fluorescent probes can help inaccurate diagnosis. Hydrogen sulfide (H S) has been reported to be involved in many physiological and pathological processes and is considered as one of the key gasotransmitters during the development of cancer. To achieve specific H S detection in cancer cells, we reported a biotin-guided NIR fluorescent sensor P1 targeting a cancer cell surface biomarker, based on the H S-specific thiolysis of the NBD-amine-hemicyanine conjugate. The probe showed a fast turn-on signal at 754 nm upon H S activation and good selectivity towards H S over millimolar levels of other biothiols. We successfully employed P1 to image endogenous H S and demonstrated its tumor-targeting ability in live cells. P1 could differentiate multiple cancer cells with various levels of H S from normal cells, indicating its potential for cancer imaging.