Valnemulin intermediate, 14-O-[(2-Amino-1, 1-dimethylethyl)thiomethylcarbonyl]mutilin, was prepared by one-pot method with Pleuromutilin, 4-toluenesulfonyl chloride and dimethylcysteamine hydrochloride in diethyl ether. The optimum condition was given following: n(Pleuromutilin): n(4-toluene sulfonyl chloride): n(dimethyl cysteamine hydrochloride)= 1:1.1:1.1, tetrabutyl ammonium bromide was used as phase transfer catalyst, amount of tetrabutyl ammonium bromide was 1.6 g, and reaction time for 2.5 h, reaction temperature for 30 ℃, the yield of product was 91%. Orthogonal design of 4 factors at 3 levels was applied for main process conditions，results agreed with the single factor experimental data. The structure of target compound was characterized by 1HNMR, IR and melting point.