Objective To investigate the optimal process parameters for the separation and purification of total flavonoids from propolis using macroporous resin combined with Sephadex LH-20 dextran gel. Methods The aluminum nitrate-potassium acetate colorimetric method was used for quantification, and the adsorption and desorption rates of nine types of macroporous adsorption resins for total flavonoids from propolis were compared to screen out the best type of macroporous adsorption resin. Dynamic adsorption and desorption tests were used to determine the optimal purification process parameters of macroporous resin, and Sephadex LH-20 dextran gel was used for further purification. Results The optimal type of macroporous resin was HPD-722 and the optimal purification process was a concentration of the loading solution of 2.5 mg/mL, a specific loading volume of 0.43 g/g (medicinal material/wet resin), a loading volume flow rate of 2 BV/h, and a diameter-to-height ratio was 1∶9. First, 3 BV of 30% ethanol was used to remove impurities, and then 4 BV of 80% ethanol was used for elution, with a volume flow rate of 1 BV/h. The eluate was concentrated and dried to obtain total flavonoids from propolis Ⅰ with a purity of 41.42%, and then ultrasonic dissolution with a small amount of methanol was performed for 1.2 g of the total flavonoids from propolis Ⅰ, which was loaded to a Sephadex LH-20 column and eluted with methanol at a flow rate of 0.75 mL/min, and 7.5 mL was collected as a fraction. With aluminum trichloride as the chromogenic reagent, all fractions were identified by polyamide thin-layer chromatography and were then mixed and dried to obtain total flavonoids from propolis Ⅱ with a purity of 68.48%. Conclusion HPD-722 macroporous adsorbent resin combined with Sephadex LH-20 dextran gel can effectively purify the total flavonoids from propolis.