Photoacoustic (PA) imaging with much deeper tissue penetration and better spatial resolution had been widely employed for the prevention and diagnosis of many diseases. In this study, a new type of hydrogen peroxide (H₂O2)-activated photoacoustic nanoprobe [Mn-AH nanoscale coordination polymer nanodots (NCPs)] was successfully synthesized by a simple one-step method in water phase containing 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), horse radish peroxidase (HRP), and manganese ion (Mn2+). After modification by polyethylene glycol (PEG), Mn-AH NCPs exhibited excellent stability and biocompatibility for in vivo H₂O₂-responsive chromogenic assay with great specificity and sensitivity. In the presence of H₂O₂, colorless ABTS would be converted by HRP into the oxidized form with strong near-infrared (NIR) absorbance, enabling photoacoustic detection of endogenous H₂O₂. Using H₂O₂-activated Mn-AH NCPs, we have successfully performed PA imaging and H₂O₂ detection of subcutaneous murine colon CT26 tumor and deep-seated orthotopic bladder tumor. Due to the inherent Mn element existence inside the Mn-AH, this nanoprobe also serves as a good T1-weighted magnetic resonance imaging (MRI) contrast agent simultaneously. Lastly, after accomplishing its imaging functions, the Mn-AH NCPs could be cleared out from the body without any long-term toxicity, providing a new opportunity for cancer diagnosis and treatment.Photoacoustic (PA) imaging with much deeper tissue penetration and better spatial resolution had been widely employed for the prevention and diagnosis of many diseases. In this study, a new type of hydrogen peroxide (H₂O2)-activated photoacoustic nanoprobe [Mn-AH nanoscale coordination polymer nanodots (NCPs)] was successfully synthesized by a simple one-step method in water phase containing 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), horse radish peroxidase (HRP), and manganese ion (Mn2+). After modification by polyethylene glycol (PEG), Mn-AH NCPs exhibited excellent stability and biocompatibility for in vivo H₂O₂-responsive chromogenic assay with great specificity and sensitivity. In the presence of H₂O₂, colorless ABTS would be converted by HRP into the oxidized form with strong near-infrared (NIR) absorbance, enabling photoacoustic detection of endogenous H₂O₂. Using H₂O₂-activated Mn-AH NCPs, we have successfully performed PA imaging and H₂O₂ detection of subcutaneous murine colon CT26 tumor and deep-seated orthotopic bladder tumor. Due to the inherent Mn element existence inside the Mn-AH, this nanoprobe also serves as a good T1-weighted magnetic resonance imaging (MRI) contrast agent simultaneously. Lastly, after accomplishing its imaging functions, the Mn-AH NCPs could be cleared out from the body without any long-term toxicity, providing a new opportunity for cancer diagnosis and treatment.