Hydrogen　peroxide　(H2O2)　can　be　produced　in　mitochondria　and　plays　a　significant　role　in　physiological　metabolism.　Overproduction　of　H2O2　is　a　hallmark　of　many　diseases.　Therefore,　it　is　very　important　to　develop　a　highly　sensitive　method　for　detecting　H2O2　both　in　vivo　and　in　vitro.　Previously　reported　benzil-based　fluorescence　probes　are　superior　to　those　based　on　boronate　ester　in　terms　of　reaction　selectivity.　However,　the　near　infrared　(NIR)　probe　with　biocompatibility　has　been　rarely　reported　for　the　detection　of　endogenous　hydrogen　peroxide　and　the　real-time　imaging　in　biological　system.　Hemicyanine　skeleton　has　been　proven　to　be　effective　scaffold　for　NM　fluorescent　probes　for　non-invasive　optical　imaging　in　vivo.　In　this　paper,　a　Cy-H2O2　probe　for　real-time　monitoring　hydrogen　peroxide　in　organisms　was　designed　by　modifying　the　NIR　hemicyanine　framework　with　benzil　moiety.　The　results　showed　that　Cy-H2O2　exhibits　high　specificity　and　sensitivity,　and　has　good　water　solubility　and　short　response　time　(within　10　min)　for　detection　of　hydrogen　peroxide　in　vitro　and　in　vivo.　The　reaction　mechanism　was　deduced　by　detecting　product　of　the　fluorescent　probe　reacting　with　hydrogen　peroxide　using　HPLC.　The　probe　shows　a　good　linear　relationship　for　the　specific　response　to　H2O2　within　the　concentration　range　of　0-7　mu　M　and　the　detection　limit　is　65　nM.　In　addition,　the　probe　Cy-H2O2　has　been　successfully　applied　to　H2O2　detection　in　living　cells　and　zebrafish.