Abstract: OBJECTIVE To optimize the formulation of huperzine A binary ethosomes and investigate its physicochemical properties. METHODS The injection method was used to prepare huperzine A binary ethosomes. Taking lecithin content(X₁), absolute ethanol content(X₂) and mixed alcohol content(X₃) as the inspection factors, central composite design-response surface method and overall desirability were used to optimize the formulation with the encapsulation efficiency(Y₁) and average particle size(Y₂) as evaluation indicators. The physicochemical properties of ethosomes such as particle size and potential were investigated. RESULTS The optimum preparation of formulation were the lecithin content of 3.60%, the absolute ethanol content of 22.50% and the mixed alcohol content of 45.00%. Under these conditions, the average encapsulation efficiency was (94.05±0.22)%, the average particle size was (260.1±5.7)nm, the Zeta potential was (-20.9±0.3)mV and the PDI was 0.188±0.056. CONCLUSION The response equation established by central composite design-response surface method has good predictability, and can be used to optimize the formulation of huperzine A binary ethosomes. The obtained ethosomes have stable properties.