Despite considerable advancements in the treatment of colorectal cancer (CRC), the overall survival rate for patients with advanced CRC remains below 50%, primarily due to challenges posed by drug resistance and metastasis. Here, a novel "Three-in-One" Cu-based metal-organic framework nanozyme with peroxidase-like (POD-like) activity has been successfully developed, aiming to promote CRC cell death by dual targeting of oxidative stress and copper ion homeostasis, which could promote CRC cell death via apoptosis and cuproptosis, and facilitate hypoxia-inducible factor 1α (HIF-1α) degradation, leading to the reversal of chemoresistance in tumor therapy. These nanozymes, composed of copper and 2-propylimidazole (Cu-PrIm), feature a distorted Cu-N4 catalytic active center that mimics natural enzyme structures consisting of copper and histidine residues, endowing them with enzyme-like activities. The antitumor efficacy of Cu-PrIm nanozymes is validated in various in vivo models of CRC. Especially Cu-PrIm nanozymes exhibit excellent biocompatibility, biodegradability, and a tolerable toxicity profile in mouse models, making them a strong candidate for clinical translation. Taken together, the study introduces a novel therapeutic paradigm in CRC treatment by targeting these vulnerabilities and leveraging the potential using "Three-in-One" Cu-PrIm nanozymes to address multiple pathways simultaneously.