Co-N-CDs-based MXene nanocomposites (MXene@PDA/Co-N-CDs) were constructed by decorating Co-N-CDs on polydopamine-functionalized MXene nanosheets. Both Co-N-CDs and MXene nanosheets have peroxidase-like activity; when the two materials are combined to form MXene@PDA/Co-N-CDs nanocomposites, the peroxide-like activity can be further enhanced. MXene@PDA/Co-N-CDs could oxidize the substrate 3,3′5,5′-tetramethylbenziline (TMB) to form ox-TMB, as confirmed by detecting the absorption of the blue products. A highly selective colorimetric biosensor was developed for the determination of glutathione (GSH) in the concentration range of 0.3 to 20 µM with a lower detection limit (LOD) of 0.12 µM, which realized the accurate detection of GSH in human serum and urine samples. Moreover, in the tumor microenvironment, MXene@PDA/Co-N-CDs could catalyze hydrogen peroxide to produce hydroxyl free radicals and produce a photothermal effect under the exposure of NIR-I irradiation. The catalytic activity of MXene@PDA/Co-N-CD nanocomposites was fully achieved for the death of cancer cells through photothermal/photodynamic synergistic therapy. The MXene@PDA/Co-N-CDs nanozyme offers multiple applications in GSH detection and tumor therapy.