•J. regia polyphenols-H. serotina polysaccharides nanoparticles were constructed.•The physicochemical properties of JRP-HSP NPs were systemically elucidated.•JRP-HSP NPs possessed sustain-release effect under gastrointestinal digestion.•The bioavailability of JRP was remarkably improved. In order to improve the bioavailability of Juglans regia L. polyphenols (JRP) in human body, the novel Juglans regia L. polyphenols-Hohenbuehelia serotina polysaccharides (HSP) nanoparticles (JRP-HSP NPs) were synthesized based on electrostatic interaction. Moreover, the physicochemical properties, morphologies and gastrointestinal digestive characteristics of JRP-HSP NPs were respectively measured and analyzed. Among four kinds of JRP-HSP NPs prepared by different condition of mass ratio, JRP-HSP NPs (1:16) with encapsulation efficiency of 51.62%, average particle size of 341.0 nm, and zeta potential of −29.3 mv, presented better physicochemical properties, such as thermal stability, photostability and crystalline properties. Morphological analysis revealed JRP-HSP NPs (1:16) appeared spherical and almost uniform. Through evaluation of simulated gastrointestinal digestion, JRP-HSP NPs prevented the variation of phenolic composition induced by gastrointestinal digestion, which was similar as the one of undigested JRP, while un-encapsulated JRP exhibited totally different state due to degradation and biotransformation, suggesting that JRP-HSP NPs possessed the sustained release characteristics in gastrointestinal system. This study provided a non-toxic strategy to encapsulate polyphenols for improving their bioavailability.