Abstract: Usnic acid (UA) has excellent antibacterial activity, but its poor water solubility and low bioavailability greatly limit its clinical application. In this study, polycaprolactone-polyethylene glycol (PCL-PEG) polymer material was used to construct UA micelles by film dispersion method. Single-factor test and Box-Behnken response surface method were used to optimize the preparation conditions, so as to improve the water solubility and bioavailability of UA. The particle size, zeta potential, microstructure, embedding effect, water solubility, drug release in vitro, stability and safety of UA micelles were characterized. The antibacterial activity of UA micelles against four common bacteria in vitro and the therapeutic effect on Staphylococcus aureus infected mice in vivo were investigated. The pharmacokinetic characteristics of UA micelles in rats were clarified. The results show that UA micelles are uniform size and smooth surface spheres with a particle size of 120.4 nm, zeta potential of −7.8 mV, encapsulation rate and drug loading of 80.66% and 9.47%, respectively. UA is successfully embedded in the hydrophobic core of the micelles, and the water solubility is increased by 19.11 times. UA micelles have a certain slow-release performance, and the cumulative drug release rate is 76.26% after 48 h. After 60 days of storage at 4℃, it shows good stability and good drug safety, and has certain antibacterial activity against many common bacteria. In addition, the therapeutic effect of UA micelles on Staphylococcus aureus infected mice is better than that of the original UA drug. Compared with the original UA drug, the relative bioavailability of UA micelles in rats is improved by 156%, C_max of UA micelles is increased by 1.34 times, the half-life of UA micelles is extended by 21.38%, and clearance rate of UA micelles is decreased by 32.39%. This study can provide a theoretical basis for the clinical application of UA micelles.