The separation and purification conditions of total flavonoids from Flos Lonicerae were studied by polyamide column chromatography. Based on the concentration of the total flavonoids, by the way of the adsorption and desorption abilities of polyamide resin, the optimum parameters of the separation and purification of the total flavonoids of Hibiscus manihot L are selected and evaluated. The main results are as follows: Purification conditions are as follows: the concentration of sample solution is 4.68 mg/mL,the pH is 4~5, adsorption rare is 1.5 mL/min, sample volume is 1.5 BV, water washing resin column with 3.0 BV, the elution rate is 1.5 mL/min with 5.0 BV 80% ethanol has a best effect with polyamides for five times. The result provided a basis for further development andapplication of Hibiscus manihot L.