Background:　Inflammatory　bowel　disease　(IBD)　is　a　prevalent　inflammatory　disorder,　and　the　abnormal　expression　of　nitric　oxide　(NO)　produced　by　biocatalysis　of　iNOS　enzyme　in　mitochondria　is　directly　associated　with　the　occurrence　and　progression　of　IBD.　Activatable　fluorescent　probes　offer　promising　tools　for　early　diagnosis　of　IBD,　however,　inadequate　biodistribution　and　limited　targeting　properties　of　these　probes　in　vivo　severely　impede　accurate　diagnosis　of　IBD　and　real-time　evaluation　of　inflammatory　levels　in　situ.　Therefore,　it　is　necessary　to　design　a　highly　efficient　fluorescent　probe　towards　NO　to　overcome　inadequate　biodistribution　and　achieve　accurate　diagnosis　and　evaluation　of　IBD　in　situ.　Results:　We　designed　a　highly　efficient　mitochondria-targeted　“turn-on”　NIR　fluorescent　probe　Cy-OMe　which　has　excellent　targeting　properties　and　imaging　ability.　The　response　mechanism　is　probe　Cy-OMe　rapidly　undergoes　N-nitrosation　reaction　resulting　in　“turn-on”　NIR　fluorescence　signal　when　exposed　to　NO.　Cy-OMe　exhibits　high　sensitivity　and　specificity　in　detecting　NO　content　in　vitro,　owing　to　its　large　Stokes　shift.　Furthermore,　the　probe　Cy-OMe　not　only　efficiently　targets　mitochondria　but　also　enables　precise　assessment　of　fluctuations　in　endogenous　NO　concertation　across　various　cell　types.　Importantly,　by　virtue　of　large　Stokes　shift　and　excellent　mitochondrial　targeting　ability,　Cy-OMe　has　the　capability　to　specifically　evaluate　dynamic　fluctuations　of　NO　in　lipopolysaccharide　(LPS)‐stimulated　IBD　mouse　models　in　situ　and　Cy-OMe　was　achieved　high-contrast　imaging　and　precision　diagnosis　of　intestinal　inflammation　diseases.　Significance:　Cy-OMe　can　accurately　assess　fluctuations　in　NO　levels　and　show　high　signal　fidelity　in　the　diseased　intestine　region,　which　has　prospects　in　the　non-invasive　diagnosis　of　intestinal　inflammation　in　vivo.　At　the　same　time,　it　is　expected　to　serve　as　a　potential　diagnose　platform　for　investigating　the　physiological　processes　underlying　NO-related　inflammatory　diseases　and　promoting　understanding　of　the　pathological　functions　of　NO　across　diverse　inflammatory　diseases.　©　2024　Elsevier　B.V.