Abstract: To optimize conditions for extracting and antibacterial activity of berberine from the fruit of Berberis thunbergii var atropurpurea, the single factor and orthogonal test were used. The inhibitory effects of berberine on six plant bacteria (Fusarium solani, Botrytis cinerea, Fusarium oxysporum, Colletotrichum gloeosporioides, Magnaporthe grisea and Cytospora sp) were determined by plate bacteriostatic tests. The antibacterial activities of the compatibility of Radix Puerariae and Berberis thunbergii var atropurpurea, and pure Berberis thunbergii var atropurpurea against Escherichia coli and Staphylococcus aureus were evaluated via the filter paper disk. The optimal extraction conditions for berberine were 120 mg of cellulase, 120 min enzymolysis time, material-liquid ratio 1:12 g·mL^-1, extraction time 150 min. The extraction rate reached 0.198%. The order of antibacterial activity of six plant pathogens was as follows:Fusarium oxysporum > Cytospora sp > Botrytis cinerea > Magnaporthe grisea > Fusarium solani > Colletotrichum gloeosporioides. Berberine had better antibacterial effect on Fusarium oxysporum. Bacteriostatic rate reached 89.28% when the content of berberine was 4 mg/mL. The combination of Berberis purpurea and Pueraria lobata had strong antibacterial activity against Staphylococcus aureus, and the best compatibility for inhibiting Escherichia coli was Pueraria lobata:Purple Berberis purpurea mass ratio 6:44. In summary, the extraction process obtained in this experiment is stable and feasible, and it can be used for the extraction of berberine from fruit of Berberis thunbergii var atropurpurea, and the berberine from purple leaf berberis has strong antibacterial activity and can be used in food preservation, agricultural biological control and other fields.