Abstract: To screen an efficient fluorescent labeling method suitable for short-chain inulin (FS), thereby providing technical support and theoretical reference for investigating its interaction mechanism and labeling other polysaccharides with fluorescence. Three fluorescent labeling methods, namely fluorescein isothiocyanate-dimethyl sulfoxide/dibutyltin dilaurate (FITC-DMSO/DBTD), FITC reductive amination, and fluorescamine (FA), were employed to labeling of short-chain inulin. The properties of the labeled products were systematically characterized by determining indicators, such as degree of substitution (DS), spectral characteristics, particle size, Zeta potential, and rheological properties. All three methods successfully achieved the fluorescent labeling of short-chain inulin, but their labeling effects exhibited significant differences. The FITC-DMSO/DBTD method yielded the highest DS, which increased with increasing FITC concentration without obvious fluorescence self-quenching. Ultraviolet-visible (UV-Vis) spectroscopy and Fourier transform infrared (FT-IR) spectroscopy confirmed the successful labeling, and this method only moderately altered the structure of short-chain inulin. For the FITC reductive amination method, the DS reached a maximum when the dosage of FITC was 50 mg, whereas it decreased at high concentrations due to the fluorescence self-quenching effect. Due to the limited number of reactive groups in short-chain inulin, the FA labeling method exhibited the lowest DS, weak fluorescence intensity, and low labeling efficiency. In addition, all three methods altered the particle size, Zeta potential, and rheological properties of short-chain inulin. The FITC-DMSO/DBTD method has the advantages of high labeling efficiency, good stability, and minimal influence on the substrate structure, making it the most suitable method for the fluorescent labeling of short-chain inulin. This method provides a reliable reference for the functional research of short-chain inulin and the fluorescent labeling of other polysaccharides.