Hydrogen sulfide (H 2 S), a well-known signaling molecule, exerts significant regulatory effects on the cardiovascular and nervous systems. Therefore, monitoring the metabolism of H 2 S offers a potential mechanism to detect various diseases. In addition, biotin is significantly used as a targeting group to detect cancer cells exclusively. In this work, a biotin-guided benzoxadizole-based fluorescent probe, NP-biotin, was developed for H 2 S detection and evaluated in normal liver cell (LO2) and liver cancer cell (HepG2) lines. Results reveal that NP-biotin can detect cellular H 2 S with high sensitivity and selectivity. Moreover, NP-biotin has been confirmed to possess the ability to target cancer cells under the guidance of the biotin group.