D-Galactose (D-Gal) is a monosaccharide crucial for various physiological processes in organisms. Abnormal variations in its levels are strongly correlated with several inherited metabolic conditions, including galactosemia. Thus, creating efficient, sensitive, and accurate detection methods for D-Gal is crucial for the early diagnosis of diseases. Herein, a novel multifunctional nanozyme derived from iron-doped carbon dots (Fe-CDs) was developed, integrating intrinsic fluorescence with robust peroxidase-like properties. Galactose oxidase catalyzes the oxidation of D-Gal to generate hydrogen peroxide (H2O2). The Fe-CD nanozyme facilitates the oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB) by the resulting H2O2, ultimately producing blue oxidized TMB (oxTMB) with a distinct absorption peak at 652 nm. Simultaneously, oxTMB quenches the fluorescence of Fe-CDs at 526 nm through fluorescence resonance energy transfer, enabling dual-mode fluorescence-colorimetric detection. The limits of detection were determined to be 0.25 µM (fluorescence mode) and 19 µM (colorimetric mode), with corresponding linear ranges of 0.001–120 mM and 0.1–100 mM, respectively. Additionally, the sensing platform was successfully employed for the quantitative analysis of D-Gal in human serum samples. The fluorometric method achieved average recoveries of 95.6–102.6%, while the colorimetric method gave recoveries of 97.5–102.8%, exhibiting outstanding selectivity and reliability. This study provides a novel cascade system for D-Gal detection, highlighting its potential applications in the fields of biomedicine and biosensing.