To date, in vivo investigations of polysaccharide’s pharmacokinetics are significantly restricted by the difficulty in their detection. This study was conducted to establish the quantitative determination of Lycium barbarum polysaccharides (LBPs) based on fluorescein isothiocyanate (FITC) pre-labeling and to investigate their tissue distribution in rat. We obtained the calibration curves linear over the range of 0–25 µg/mL in rat tissue samples with correlation coefficients greater than 0.99. The inter-day and intra-day precisions (RSD, %) were within 15%, and the relative recovery ranged 95.2%–102.4%, with RSD range 1.48%–9.58%, indicating that this experiment was suitable for the determination of LBPs. The fluorescence intensity was measured after 24 h storage at room temperature, 3 times of freeze-cycle and cryopreservation at –20 °C for 15 day, these results indicated that the stability of the samples was good. LBP-FITC was mainly absorbed by the small intestine and stomach, and mainly excreted in the urine through the kidney; this distinct difference in the tissue distribution of LBPs could be attributed to the size of these LBPs in relation to the pore sizes of the vascular beds in the kidney and liver. Results showed in this study enable us to comprehensively understand the biological effects of LBPs following its oral ingestion.