Abnormal HO levels are closely related to many diseases, including inflammation and cancers. Herein, we simultaneously load HRP and its substrate, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), into liposomal nanoparticles, obtaining a Lipo@HRP&ABTS optical nanoprobe for in vivo HO-responsive chromogenic assay with great specificity and sensitivity. In the presence of HO, colorless ABTS would be converted by HRP into the oxidized form with strong near-infrared (NIR) absorbance, enabling photoacoustic detection of HO down to submicromolar concentrations. Using Lipo@HRP&ABTS as an HO-responsive nanoprobe, we could accurately detect the inflammation processes induced by LPS or bacterial infection in which HO is generated. Meanwhile, upon systemic administration of this nanoprobe we realize in vivo photoacoustic imaging of small s.c. tumors (∼2 mm in size) as well as orthotopic brain gliomas, by detecting HO produced by tumor cells. Interestingly, local injection of Lipo@HRP&ABTS further enables differentiation of metastatic lymph nodes from those nonmetastatic ones, based on their difference in HO contents. Moreover, using the HO-dependent strong NIR absorbance of Lipo@HRP&ABTS, tumor-specific photothermal therapy is also achieved. This work thus develops a sensitive HO-responsive optical nanoprobe useful not only for in vivo detection of inflammation but also for tumor-specific theranostic applications.