Herein, we report a novel type of NPs by loading paeonol (Pae) into PLGA NPs, to enhance drug stability and oral bioavailability. The paeonol (Pae)-loaded polylactic-co-Gly-colic acid (PLGA) nanoparticles (Pae-PLGA-NPs) were prepared by nanoprecipitation method. The resultant NPs were in spherical shape with an average particle size around 237.7 ± 4.92 nm, and the PDI and zeta potential were 0.110 ± 0.01 and −25.33 ± 1.37 mV, respectively. The encapsulation efficiency (EE) and drug loading (DL) of the Pae-PLGA-NPs were 86.26 ± 1.12 and 12.74 ± 0.37% respectively. The in vitro drug release, in vivo pharmacokinetics and in situ single-pass intestinal perfusion (SPIPs) of Pae-PLGA-NPs was investigated. In vivo, the AUC_(0-t), C _max, MRT_(0-t), and T_1/2z of the Pae-PLGA-NPs group were 3.79-, 1.89-, 1.40- and 1.49-fold greater than those of the Pae suspension group, respectively. The in situ single-pass intestinal perfusion of NPs results showed the Ka values in the duodenum, jejunum, ileum and colon were 1.12-, 1.40-, 1.52- and 2.21-fold higher than those of Pae solution, respectively. Moreover, the Papp values of the ileum and colon were 1.27- and 1.31-fold higher than those of the solution group. Such findings suggested the Pae-PLGA-NPs can significantly improve the intestinal absorption characteristics, and have a beneficial effect on oral administration as a nanometer-sized carrier.