Taking the adsorption rate and desorption rate as the evaluation indexes, macroporous resin was adopted to optimize the separation and purification technology of total flavonoids from Catalpa root bark, and the antibacterial activity of the purified total flavonoids from Catalpa root bark was investigated. The results indicated that the optimal technological conditions for the separation and purification of total flavonoids from Catalpa root bark were as follows: application of NKA-II resin; mass concentration, flow rate and pH value of loading buffer of 0.538 mg/mL, 1.66 mL/min and 4.01, respectively; concentration of eluant ethyl alcohol of 70%; and elution flow rate of 2.5 mL/min. Under such conditions, the purity, adsorption rate and desorption rate of total flavonoids from Catalpa root bark were (77.43±0.23)%, (93.36±0.25)% and (95.51±0.17)%, respectively. Antibacterial activity indicated that total flavonoids from Catalpa root bark had extremely sensitive antibacterial activity to Escherichia coli, Bacillus subtilis, and Staphylococcus aureus; and high antibacterial sensitivity to penicillium and Saccharomyces cerevisiae. MIC values of S. aureus, Bacillus subtilis and E. coli, as well as S. cerevisiae and penicillium were (6.25±0.25) mg/mL, (12.50±0.36) mg/mL and (25.00±0.27) mg/mL, respectively.