Abstract: Longan is one of the most valuable subtropical fruits in Southeast Asian regions. This fruit can also play an important role in the blood metabolism for neuropathic pain and swelling. Many studies have focused on the functional activity of longan polysaccharides (LP) in recent years. However, it is still unclear on the structural characteristics, as well as the absorption and distribution in vivo of LP. In this work, liquid chromatography, ion chromatography, and nuclear magnetic resonance spectroscopy (NMR) were employed to investigate the structure of LP. The results showed that the LP consisted of the glucose (Glc), fructose (Fru), arabinose (Ara), galactose (Gal), galacturonic acid (GalA), xylose (Xyl), mannose (Man) and rhamnose (Rha) with the molar ratios of 28.94%, 27.63%, 19.13%, 8.01%, 6.71%, 5.32%, 2.40%, and 1.85%, respectively. The Two-dimensional (2D) NMR spectra of HSQC, COSY, and HMBC revealed that the LP shared the following chemical shifts: δ, 5.00/101.59, 4.55/97.40, 5.13/100.04, 4.47/96.11, 3.62/63.56, and 5.15/109.17, respectively. Moreover, these peaks were attributed to the H-1/C-1 signals of the A-F sugar residues. Ultimately, the LP was determined as a heteropolysaccharide with 1,6)-D-Fruf-(2→5)-L-Araf-(1→4)-D-Glcp-(1→3,6)-Glcp-(1→6)-D-Galp-(1→ as the main chain. The microstructure of the LP was lamellar and stacked with a relatively smooth surface. There was the broad absorption peak near 3 445-3 440 cm⁻¹, which was attributed to O-H stretching vibrations. The broad absorption peak near 2 950-2 945 cm⁻¹ was attributed to C-H stretching vibrations. The peaks at 1 623.11 and 1 400.31 cm⁻¹ were attributed to the carboxylate ion (COO⁻). The bonds at 1 380.43 and 1 360.52 cm⁻¹ corresponded to C-O bending. The hump around 1 200-1 000 cm⁻¹ confirmed the presence of a furanose sugar. The absorption at 1 078.13 cm⁻¹ was related to the C-O-C stretching vibrations, while the absorption peaks around 938-842 cm⁻¹ indicated the simultaneous presence of β and α configurations. In addition, the characteristic peaks at 1 660 cm⁻¹ and 1 580 cm^-1 were found in the FTIR spectrum of LP-Cy5.5, which belonged to the N-H bending vibrational bands of amide I and amide II, respectively. Meanwhile, the signals at 6.5-8.0 ppm belonged to the benzene ring of Cy5.5, compared with the 1H NMR of LP. As such, the successful synthesis of LP-Cy5.5 was realized after characterization. In addition, the LP with the near-infrared fluorescent dye Cy5.5 was selected as a fluorescent marker to assess the digestion and distribution of LP in mice. In vitro intestinal imaging revealed that there were fluorescent signals in all intestinal segments at the early stage. The fluorescence signal of LP-Cy5.5 was mainly distributed in the abdomen of mice. Particularly, the strong fluorescent signals of the LP-Cy5.5 were observed in the stomach, small intestine, cecum, and colon during 1-4 h of digestion, and then gradually disappeared afterward. It indicated that the LP was partially absorbed by the small intestine and then entered the large intestine, where it was utilized by the intestinal microbes. This finding can also provide a fundamental basis for the development of functional foods containing longan polysaccharides. Meanwhile, it is of great significance to obtain the value-added and efficient utilization of resources in order to promote the sustainable longan industry.