Abstract: [objective] to optimize the extraction and determination method of the total flavonoids thus to screen the cell clones and plantlets with high contents, [method] the ultrasonic technique was used and several treatments were tried and screened, [result] the best condition was marinated 6 hours with 75% carbinol then ultrasonic extracted twice. And then the content was determined with colorimetry from cultures of Glycyrrhiza uralensis Fisch. The result showed the recovery rate was between 95.3% and 101.4% and the RSD was 1.61%. The total flavonoids of the initial calli was between 0.0012% and 0.05%, the improvement cotyledonary calli was up to 0.08%, the contents of the plantlets were between 1.04% and 1.31%, and after being transplanted was up to 4.5%. [conclusion] The rate of veracity of this method was high and this provided the determination of Glycyrrhiza cultures flavonoids products a feasible method, and those data offered a credible academic gist for authenticate of the experiment resource.