Abstract: Three flavonoid compounds(quercetin, kaempferol and isorhamnetin)in Anoectochilus roxburghii cultivated with four different modes were determined by HPLC. The above metioned compounds were extracted by ultrasonic-microwave synergistic extraction method and the quantitative and qualitative analysis were separately analyzed by HPLC and HPLC-ESI-MS. Quercetin, kaempferol and isorhamnetin had good linear relationship in the range of 0.25~20.0(r=0.999 2), 0.25~20.0(r=0.999 5), and 1.0~80.0(r=0.999 0), respectively. The contents were within 151.8~240.0、47.2~88.2、16.8~29.8 μg/g, respectively. The method is a simple, accurate and fast method for the quality evaluation of A. roxburghii cultivated with different modes. The contents of three flavonoid compounds increased effectively in A. roxburghii with the addition of nitrogen, phosphorus, potassium(NPK) compound fertilizer in cultivation and decreased 2.3%, 17.2% and 18.8% respectively in A. roxburghii with the addition sulfur-phosphorus(SP) compound fertilizer, this finding provides scientific guidance for planting A. roxburghii.