A photooxidation strategy was used to develop near-infrared (NIR) afterglow carbon nanodots (CDs) with an ultralong lifetime of up to 5.9 hours, comparable to rare-earth or organic long-persistent luminescent materials. The CDs exhibit size-dependent afterglow lifetimes ranging from 3.4 to 5.9 hours. Structural and ultrafast dynamics analysis, along with density functional theory (DFT) simulations, revealed that photooxidation-induced dioxetane intermediates activate persistent luminescence by slowly releasing energy via steric hindrance effects. The CDs achieve a tissue penetration depth of 20 mm and demonstrate high signal-to-background ratio, biological safety, and cancer-specific targeting, enabling ultralong-afterglow guided surgery in mice models for accurate tumor removal. These results highlight the potential of CDs for precision tumor resection. The persistent luminescence of CDs is activated by photooxidation-induced dioxetane intermediates, which slowly release energy via steric hindrance effects. The CDs' afterglow emission is not quenched by dissolved oxygen, making them suitable for biological imaging. The CDs have tunable wavelength, high physicochemical inertness, low biological toxicity, and easy preparation, making them promising for biological imaging, information encryption, and light-emitting devices. The CDs' afterglow emission is enhanced by photooxidation, which increases their afterglow lifetime and stability. The CDs' afterglow emission is not quenched by dissolved oxygen, making them suitable for biological imaging. The CDs have a high signal-to-background ratio, good biosafety, and effective tumor targeting, enabling ultralong-afterglow guided surgery in mice models for accurate tumor removal. These results demonstrate the potential of CDs for precision tumor resection.A photooxidation strategy was used to develop near-infrared (NIR) afterglow carbon nanodots (CDs) with an ultralong lifetime of up to 5.9 hours, comparable to rare-earth or organic long-persistent luminescent materials. The CDs exhibit size-dependent afterglow lifetimes ranging from 3.4 to 5.9 hours. Structural and ultrafast dynamics analysis, along with density functional theory (DFT) simulations, revealed that photooxidation-induced dioxetane intermediates activate persistent luminescence by slowly releasing energy via steric hindrance effects. The CDs achieve a tissue penetration depth of 20 mm and demonstrate high signal-to-background ratio, biological safety, and cancer-specific targeting, enabling ultralong-afterglow guided surgery in mice models for accurate tumor removal. These results highlight the potential of CDs for precision tumor resection. The persistent luminescence of CDs is activated by photooxidation-induced dioxetane intermediates, which slowly release energy via steric hindrance effects. The CDs' afterglow emission is not quenched by dissolved oxygen, making them suitable for biological imaging. The CDs have tunable wavelength, high physicochemical inertness, low biological toxicity, and easy preparation, making them promising for biological imaging, information encryption, and light-emitting devices. The CDs' afterglow emission is enhanced by photooxidation, which increases their afterglow lifetime and stability. The CDs' afterglow emission is not quenched by dissolved oxygen, making them suitable for biological imaging. The CDs have a high signal-to-background ratio, good biosafety, and effective tumor targeting, enabling ultralong-afterglow guided surgery in mice models for accurate tumor removal. These results demonstrate the potential of CDs for precision tumor resection.