As nanozymes, carbon dots (CDs) have attracted increasing attention due to their remarkable properties. Besides general enzyme activity, their photoluminescence and photothermal properties have been explored rarely, whereas their synergistic effects might produce CDs-based nanozymes of high performance. Here, iron-doped CDs (Fe-CDs) with tunable fluorescence and enhanced peroxidase-like activity were designed to develop a novel "three-in-one" multifunctional platform to provide dual-mode/dual-target detection and near infrared (NIR)-assisted antibacterial ability. This proposed strategy for a H2O2 test exhibited a wide linear relationship with a low limit of detection (LOD) of 0.16 μM (colorimetric) and 0.14 μM (ratiometric fluorescent). Furthermore, due to the nature of cholesterol being oxidized to H2O2 by cholesterol oxidase, sensitive and selective detection of cholesterol was realized, and the LOD was 0.42 μM (colorimetric) and 0.27 μM (ratiometric fluorescent), surpassing that reported previously. This result suggested that Fe-CDs could be used for dual-mode quantification of large family of H2O2-producing metabolites, thereby paving the way for developing multi-mode sensing strategies based on nanozymes. Moreover, this platform showed synergistic effects for antibacterial application, indicating great prospects for bacterial killing as well as wound disinfection and healing. Hence, this platform could contribute to the construction of multifunctional CDs with high performance.