To determine the content of epimedium flavonoids in Erxian Decoction by high performance liquid chromatography(HPLC),and to analyze Herba Epimedii from 4 different habitats.Methods：Sample volume was 1 μL; column temperature was controlled at 40 ℃; mobile phase A was homogeneously mixed with 0.1% pure formic acid and 5 mmol/L pure ammonium acetate; mobile phase B was homogeneously mixed with pure methanol and ultra-pure water.Linear gradient elution procedures of high performance liquid chromatography were studied.When the detection wavelength was set at 270 nm,more absorption peaks can be detected.In the early stage,the routine reliability methodologies of HPLC were investigated,including specificity,precision,stability,etc.Then the contents of icariin,icariin II,Icariin B,Icariin C were further determined and analyzed.Results：1）Icariin,Icariin II,Chaohoding B and Chaohoding C in Erxian Decoction showed good isolation,and there was no endogenous interfering substance.Each flavonoid had good specificity. 2）The results of linear regression analysis showed that the linear regression equation of icariin was Y=34.012X-12.99; the linear regression equation of icariin II was Y=39.875X-7.134; the linear regression equation of Chaohuoding B was Y=23.997X-8.416; and the linear regression equation of Chaohuoding C was Y=29.896X-15.38. 3）The RSD values of precision tests of icariin,icariin II,Chaixiding B,Chaixiding B,and Chaixiding C were 0.33%,0.64%,0.57%,0.52%.RSD values of stability test were 1.09%,1.62%,1.78%,0.41%.RSD values of repetitive test were 2.61%,2.75%,2.48%,1.22%. 4）The average recoveries of icariin,icariin II,Chaohoding B and Chaohoding C were(96.87±2.15)%,(99.46±1.52)%,(101.64±2.43)%,(99.79±2.31)%,respectively. 5）The content of epimedium flavonoids in Erxian Decoction was higher than that of sample solution of single herb of Herba Epimedii.From the point of view of the content of flavonoids in different habitats of Herba Epimedii,it was found that the content of flavonoids in Herba Epimedii from Sichuan was higher than that in the other 3 habitats,followed by Herba Epimedii from Shaanxi,Herba Epimedii from Hubei,and Herba Epimedii from Hunan.The content of flavonoids was the lowest.Conclusion：The determination of flavonoids in Erxian Decoction by HPLC has good reliability,and the content of flavonoids in Erxian Decoction is higher than that of single herb of Herba Epimedii.