Sequential column chromatographies on HPD-100 macroporous adsorption resin (MAR) and polyamide (PA) were used to purify total flavonoids from Ampelopsis grossedentata. The optimum adsorption parameters for HPD-100 MAR were determined as follows: sample concentration, 6 mg/mL; sample flow rate, 1 mL/min; and sample volume, 130 mL. Under these conditions, the percentage adsorption was 97.14%. The optimum desorption parameters were established as follows: eluent concentration, aqueous ethanol (70:30, V/V); eluent volume, 40 mL; and flow rate, 1 mL/min. Under these conditions, the percentage desorption was 94.10%. After the treatment with HPD-100 MAR, the purity of total flavonoids was increased from 55.00% to 72.25%. The optimum adsorption parameters for PA were determined as follows: sample concentration, 6 mg/mL; and flow rate, 2 mL/min. Under these conditions, the percentage adsorption was 99.57%. The optimum desorption parameters were determined as follows: 70% ethanol in water (V/V) as the eluent; eluent volume, 55 mL; and flow rate, 1 mL/min. Under these conditions, the percentage desorption was 76.50%. After the treatment with PA, the purity of total flavonoids was further increased to 80.75%. The method presented in this study can provided a promising approach for the efficiency purification of total flavonoids from Ampelopsis grossedentata. © 2016, China Food Publishing Company. All right reserved.