High surface area activated carbon was prepared from waste tobacco after extracting nicotine with microbial enzymatic and chemical activation. Surface properties of the prepared carbons were performed using nitrogen adsorption, and the adsorption behavior of the prepared carbons under different operation conditions on methyl orange was investigated by a batch adsorption experiment. The experimental results show that: using waste tobacco 5.0035g and white- rot fungi volume is 2.0ml, under the conditions of enzymatic time of 36h, activation temperature at 600˚C and activation time of 2h, the concentration of ZnCl2 activation is 20%,The BET surface area of carbons prepared reach 1356.53m2/g, the average aperture is3.78nm, and the hole dimension is0.17 ml/g. The adsorption amount on methyl orange of 43mg/L reach 4979.31mg/g, and it show high adsorption capacity.