The optimal preparation conditions of sea buckthorn oil-poly (lactic-co-glycolic acid) (PLGA) microspheres were studied via the emulsion-solvent evaporation process coupling ultrasonic treatment, using polyvinyl alcohol (PVA) as the emulsifier, PLGA as the wall material, a mixture of dichloromethane and ethyl acetate as the oil phase. The research discussed the effects of the PVA concentration, the PLGA dosage, ultrasonic power and ultrasonic time on comprehensive quality index (S) of sea buckthorn oil-PLGA microspheres. The orthogonal experimental design L9 (34) with the factor analysis was applied to optimize preparation conditions. The experimental date showed that the optimal technology preparation conditions for sea buckthorn oil-PLGA microspheres were as follows: the concentration of PVA 0.125 g/ml, PLGA loading 125 mg, ultrasonic time 10 min, and ultrasonic power 180W. It resulted in sea buckthorn oil-PLGA microspheres that were in more regular and smaller shape, and with a better comprehensive quality index.