Nanoparticles based on Radix pseudostellariae protein-polysaccharide conjugates were self-assembled via pH adjustment and thermal treatment. The fabricated nanoparticles (CP3) were spherical with narrow size distribution of 125.0 nm in diameter. The doxorubicin (DOX) -loaded CP3 nanoparticles exhibited pH-sensitive release behavior and accelerated the release of DOX under the acidic pH simulating tumor microenvironment and endosomal pH. In HepG2 uptake studies, CP3-DOX nanoparticles notably improved the internalization of DOX, which was 1.56-fold compared with free DOX. CP3-DOX nanoparticles could serve as P-glycoprotein efflux pump inhibitor and be internalized into HepG2 cells via clathrin-dependent endocytosis. Moreover, the cytotoxicity effect of DOX on HepG2 cells was elevated after the encapsulation by CP3, with a lower IC50 value of 0.25 μg/mL. The findings suggested that the pH-sensitive CP3-DOX nanoparticles has a great potential in facilitating the efficacy of DOX in cancer cells, and the obtained CP3 could be a good candidate as nanocarrier for the encapsulation and delivery of functional compounds.