Oral administration represents the primary route for administering polysaccharides. Pharmacokinetics serves as a critical bridge between the administration of polysaccharides and the manifestation of their bioactivities. However, the pharmacokinetic behavior of polysaccharides remains poorly understood. This study aimed to investigate the in vivo pharmacokinetics of an orally administered Ruditapes philippinarum polysaccharide (ERPP), including its plasma concentration-time profile, tissue distribution, and excretion. Moreover, a sensitive quantitative method was developed using 5-DTAF as a fluorescent probe to quantify ERPP levels in rat plasma and tissues. The results revealed that ERPP was absorbed in the gut (Tmax = 3 h), reaching a peak concentration (Cmax) of 22.7 ± 2.7 mg/L. Subsequently, it predominantly accumulated in the kidneys, liver, and lungs. Complementarily, near-infrared fluorescence (NIR) imaging provided real-time qualitative visualization of ERPP-Cy5.5 tissue distribution, corroborating the organ-level accumulation pattern observed in the quantitative study. Excretion studies over a 24 h observation window indicated that ERPP was primarily excreted via feces, with 45.7 ± 4.4% of the recovered dose within this period; the complete excretion profile warrants further investigation at extended time points. These findings provide pharmacokinetic insights into the in vivo fate of marine polysaccharides and support further biological evaluation.