Acetylpuerarin (AP), an acetylated derivative of puerarin, shows brain-protective effects in animals. However, AP has low oral bioavailability because of its poor water solubility. The objective of this study was to design and develop poly(lactide- co-glycolide) (PLGA) nanoparticles (NPs) to enhance the oral bioavailability of AP. The NPs were prepared using a solvent diffusion method optimized via uniform design (UD) combined with response surface methodology (RSM) and characterized by their morphology, particle size, zeta (ζ)-potential, encapsulation efficiency (EE), drug loading (DL), and in vitro drug release. A pharmacokinetic study was conducted in Wistar rats administered a single oral dose of 30 mg/kg AP. The optimized NPs were spherical and uniform in shape, with an average particle size of 145.0 nm, a polydispersity index (PI) of 0.153, and a ζ-potential of −14.81 mV. The release of AP from the PLGA NPs showed an initial burst release followed by a sustained release, following Higuchi’s model. The EE and DL determined in the experiments were 90.51% and 17.07%, respectively. The area under the plasma concentration-time curve (AUC 0−∞) of AP-PLGA-NPs was 6,175.66±350.31 h ng/mL, which was 2.75 times greater than that obtained from an AP suspension. This study showed that PLGA NPs can significantly enhance the oral bioavailability of AP.