Abstract: The residue dynamic of difenoconazole, carbendazim and dimethomorph in fresh plant of Dendrobium officinale and its growth media were investigated. Samples extracted with acetonitrile were purified by solid-phase dispersion method and analysed by HPLC-MS/MS. The response surface method was used to modify the best cleaning mode which involved the washing time and the solution concentration (NaCl and NaHCO3). The results showed that the average recoveries of 3 commonly used pesticides were 78.5%-106% and RSDs were 0.7%-4.5% at different spiking concentrations, which meets the inspection requirement of pesticide residues. When recommended dosage and double recommended dosage of pesticides were applied, the half-lives of difenoconazole, carbendazim and dimethomorph were 3.8-3.9 d, 2.6 d and 2.8-2.9 d in fresh plant and 4.1-5.6 d, 2.8-3.1 d and 3.6-5.1 d in the growth media, respectively. The optimal cleaning conditions for difenoconazole were water washing for 45 min after washing in the mixture of 5.49% NaCl and 1.27% NaHCO3 for 5 min. As for carbendazim, the conditions were water washing for 45 min after washing in the mixture of 5.34% NaCl and 1.22% NaHCO3 for 5 min. And the conditions for dimethomorph were water washing for 59 min after washing in the mixture of 2.07% NaCl and 0.80% NaHCO3 for 5 min. This study provided stable and excellent basis for the establishment of D. officinale safety assessment system.