Abstract: To prepare and optimize luteolin nanostructured lipid carriers (Lut-NLCs) and investigate their antibacterial activity in vitro. Lut-NLCs were prepared by hot melt emulsification-ultrasonic method. The solid lipid concentration (X₁),liquid lipid concentration (X₂) and surfactant concentration (X₃) were used as independent variables,with the average particle size (Y₁) and the encapsulation efficiency (Y₂) as the dependent variables. The optimal formulation of Lut-NLCs was obtained through Box-Behnken experiment design. The microstructure of Lut-NLCs was observed by transmission electron microscopy(TEM). The in vitro release characteristics of Lut-NLCs were investigated. Furthermore, the in vitro antibacterial activities of luteolin and Lut-NLCs were compared. The formulation composition of Lut-NLCs was optimized as follows:the concentration of the solid lipid, liquid lipid and surfactant were 13.0 mg/mL,15.0 mg/mL,and 15.0 mg/mL,respectively. Three batches of Lut-NLCs were prepared with an average particle size of (210.4±17.3) nm,and an encapsulation efficiency of (88.4±1.2)%. Lut-NLCs were observed to be spheroidal,with a smooth surface and a uniform particle size distribution by TEM. The drug release profiles of Lut-NLCs showed a bursting release in the early stage and a slow and stable release in the later stage. Moreover, the cumulative release amount of drug reached 95% in 12 hours. The results of antibacterial circle experiment showed that the antibacterial effect of Lut-NLCs on Staphylococcus aureus and Escherichia coli was higher than that of luteolin raw materials. In this study,the formulation of Lut-NLCs prepared by simple preparation process is reasonable,and Lut-NLCs also exhibited the significant in vitro antibacterial activity. It is expected to be an effective way for external application of luteolin.