Benefiting from the advantages like large surface area, flexible constitution, and diverse structure, metal-organic frameworks (MOFs) have been one of the most ideal candidates for nanozymes. In this study, a nitro-functionalized MOF, namely NO₂-MIL-53(Cu), was synthesized. Multi-enzyme mimetic activities were discovered on this MOF, including peroxidase-like, oxidase-like, and laccase-like activity. Compared to the non-functional counterpart (MIL-53(Cu)), NO₂-MIL-53(Cu) displayed superior enzyme mimetic activities, indicating a positive role of the nitro group in the MOF. Subsequently, the effects of reaction conditions on enzyme mimetic activities were investigated. Remarkably, NO₂-MIL-53(Cu) exhibited excellent peroxidase-like activity even at neutral pH. Based on this finding, a simple colorimetric sensing platform was developed for the detection of H₂O₂ and glucose, respectively. The detection liner range for H₂O₂ is 1–800 μM with a detection limit of 0.69 μM. The detection liner range for glucose is linear range 0.5–300 μM with a detection limit of 2.6 μM. Therefore, this work not only provides an applicable colorimetric platform for glucose detection in a physiological environment, but also offers guidance for the rational design of efficient nanozymes with multi-enzyme mimetic activities.