Inflammatory bowel disease (IBD) is a chronic and relapsing disease of the gastrointestinal tract. At present, antioxidant therapy is a promising strategy for IBD treatment. Since low-molecular-weight antioxidants (e.g., 2,2,6,6-tetramethylpiperidin-N-oxyl (TEMPO)) have a short in vivo half-life and inadequate cellular uptake, researchers have focused on loading antioxidants into nanostructures for improving their antioxidant and anti-inflammatory activities. As we know, in situ self-assembly with the formation of nanostructures under intracellular specific stimuli is a convenient delivery strategy to enhance the accumulation and retention of molecules at target sites in vivo. Herein, we developed an in situ self-assembled TEMPO probe TPP-FFYp-O to improve the antioxidant and anti-inflammatory effects of TEMPO in IBD. Compared to the control probe TPP-O without a self-assembly moiety, TPP-FFYp-O could successfully self-assemble into nanoparticles (NPs) under alkaline phosphatase (ALP)-guided dephosphorylation, with significantly enhanced antioxidant capacity in vitro. Cell experiments confirmed that intracellular formation of NPs by TPP-FFYp-O could improve the antioxidant and anti-inflammatory abilities of TEMPO and alleviate cellular damage. Moreover, TPP-FFYp-O exhibited good biocompatibility in vivo and significantly relieved pathological injury and inflammatory factors in the colon tissues of an IBD model compared to TPP-O. We envision that the in situ self-assembly platform can be used to load various active molecules for more applications in the future.