Imaging cancer specific biomarkers with near-infrared (NIR) fluorescent probes can help inaccurate diagnosis. Hydrogen sulfide (H₂S) 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 H₂S detection in cancer cells, we reported a biotin-guided NIR fluorescent sensor P1 targeting a cancer cell surface biomarker, based on the H₂S-specific thiolysis of the NBD–amine–hemicyanine conjugate. The probe showed a fast turn-on signal at 754 nm upon H₂S activation and good selectivity towards H₂S over millimolar levels of other biothiols. We successfully employed P1 to image endogenous H₂S and demonstrated its tumor-targeting ability in live cells. P1 could differentiate multiple cancer cells with various levels of H₂S from normal cells, indicating its potential for cancer imaging.