OBJECTIVE: To prepare Usnic acid self-microemulsion, prescription optimization and evaluate its quality. METHODS: Usnic acid self-microemulsion was prepared by emulsification method using medium chain triglycerides-single linoleic acid glyceride (1 ∶ 1, m/m) as oil phase, polyoxyethylene hydrogenated castor oil as emulsifier, diethylene glycol monoethyl ether as co-emulsifier. Based on the solubility test and pseudotemary phase diagram, using self-emulsifying time (t), light transmittance (T), drug equilibrium solubility (S), accumulative dissolution rate at 5 min and 60 min (Q5 min, Q60 min) as evaluation indexes, central composite design-response surface methodology was utilized to optimize oil phase ratio and ratio of emulsifier to co-emulsifier (Km) in microemulsion formulation. At the same time, the optimal formulation was verified, the morphology, particle size, drug-loading amount and dissolution of Usnic acid self-microemulsion were investigated. RESULTS: The optimal formulation was as follows as oil phase ratio of 25%, Km of 2.0; ratio of mixed oil phase, emulsifier, co-emulsifier in the formulation was 25%, 50%, 25%. t, T, S, Q5 min and Q60 min of Usnic acid self-microemulsion prepared by optimal formulation were 1.96 min, 87.67%, 5.67 mg/g, 66.58%, 76.73% (all RSD<3%, n=3), respectively. The relative errors of them to predicted values were all less than 4% (n=3). Usnic acid self-microemulsion was spherical droplet in shape after diluted with water. The average particle size was 39.4 nm, average drug-loading amount was 4.55 mg/g. Q90 min of Usnic acid self-microemulsion reached 99.58% in pH 6.8 phosphate buffer (n=3). CONCLUSIONS: Prepared Usnic acid self-microemulsion is in line with quality requirements.