In this study, we have presented a novel strategy to fabricate fluorescent Fe-doped carbon dots (Fe-CDs) by one-step hydrothermal carbonization, using methylthymol blue sodium salt (MTB) and FeCl₃·6H₂O as precursors. Multiple techniques were employed to characterize the properties of the obtained Fe-CDs. Importantly, the intrinsic peroxidase-like activity of the Fe-CDs was explored. Through catalyzing the oxidization of o-phenylenediamine (OPD) by hydrogen peroxide (H₂O₂), the Fe-CDs exhibited a superior catalytic performance over horseradish peroxidase (HRP) attributed to the doped Fe elements in the Fe-CDs. By the combination of favorable fluorescence properties and excellent catalytic activities, Fe-CD based ratiometric fluorescence sensing systems toward H₂O₂ and glucose have been designed in view of the inner filter effect (IFE) between Fe-CDs and 2,3-diaminophenazine (DAP, the oxidation product of OPD). Such responses can be used for well quantifying H₂O₂ and glucose in the ranges of 0–133 μM and 0–300 μM with detection limits of 0.47 and 2.5 μM (S/N = 3), respectively. The proposed sensing system has been successfully used for the assay of glucose in human serum samples, indicating great promise as a practical platform for blood glucose sensing.