Abstract: The quantitative analysis method of high performance liquid chromatography (HPLC) was established to determine the absorption of tebuconazole by mycelium of Rhizoctonia cerealis . After the sample was extracted and purified, it was determined by the HPLC and the external standard method was used. The minimum detectable amount and concentration were 0.2 ng and 0.01 mg/kg,respectively. The recovery and precision were high. It could completely satisfy with the demands of quantitative analysis on the study of drug-fast mechanism of fungi. The method has some merits including low cost, simple and accurate, compared with traditional liquid scintillation method. The comparison of the absorption of tebuconazole was made between resistant-strains and sensitive-strain. The results showed that after treated with tebuconazole (0.5 mg/L),the amount of tebuconazole accumulated by resistant-strains was lower than that by sensitive-strain. After 24 hours, the amount of tebuconazole accumulated by sensitive-strain was 127.74% higher than that of high resistant-strain, 47.72% and 7.83% higher than that of middle resistant-strains,respectively.