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The Oenothein B (OEB) rich in eucalyptus is a kind of extremely development of prospects of antioxidant activity, but low oral bioavailability limits its application. The novel polyelectrolyte nanoparticle was designed for OEB delivery through the self-assembly of casein phosphopeptides (CPP) and natural active polysaccharides-chitosan (CS). The OEB nanoparticle with small particle size and high embedding rate were constructed to solve the problem of low oral bioavailability of OEB, and its stability as well as in vivo absorption were investigated. The storage stability analysis, digital inverted fluorescence microscopy and emission field scanning electron microscopy showed that the particle size and total amount of OEB nanoparticles did not vary significantly after 96 h of continuous storage at room temperature, and the particle size distribution was uniform, and the particles kept their integrity pH 1.2. The maximum release rate of OEB nanoparticles was only 0.72% in artificial gastric fluid and 21.77% in artificial intestinal fluid. It greatly reduces the risk of its decomposition and plays a better protective role. The results of in vivo absorption showed that OEB nanoparticle gavage mice did not detect OEB in blood and its metabolites except for OEB in gastric juice, indicating that OEB changes in vivo after release. Therefore, although OEB nanoparticles can effectively protect their decomposition in gastrointestinal fluid, it is best to achieve sustained release at the target location in order to improve the absorption and utilization effect.