Polysaccharides encounter significant challenges in vivo pharmacokinetic studies because of their complex structures and the limitations of current detection methods, thereby impeding their development and biomedical applications. This study systematically investigated the oral absorption characteristics and tissue distribution of ME-2, a homogeneous polysaccharide from Auricularia auricula-judae, using a dual-labeling pharmacokinetic approach. First, a fluorescein-5-thiosemicarbazide (FTSC)-based quantitative method was established to analyze plasma pharmacokinetics and tissue concentrations of ME-2, demonstrating robust methodological stability (intra-/inter-day RSD&#xa0;<&#xa0;15&#xa0;%) and accuracy (recovery rate 95-103&#xa0;%). Then, complementary near-infrared fluorescence (NIRF) imaging with cyanine 5.5 (Cy5.5) labeling was employed to resolve ME-2's spatial distribution in major organs. Results revealed that orally administered ME-2 achieved effective systemic absorption, with a significant accumulation in the liver (peak concentration Cmax&#xa0;=&#xa0;10.55&#xa0;&#x3bc;g/g) and lung (Cmax&#xa0;=&#xa0;11.82&#xa0;&#x3bc;g/g). Strong spatiotemporal correlations between FTSC quantification and Cy5.5 signals were observed, confirming ME-2's intrinsic oral bioavailability without structural modification. By elucidating the pharmacokinetic characteristics of ME-2, its therapeutic effect on lung diseases has been understood more deeply, thus establishing a solid scientific foundation for the development of polysaccharide-based pharmaceuticals and health supplements.