Objectives To produce and characterize novel laccases with ethanol tolerance from Trametes versicolor using agriculture by-products as energy source. Results Trametes versicolor 1017 produces two laccase isoenzymes with a total activity of 10 U ml(-1) within 8 days when using wheat bran and peanut powder as energy sources in liquid culture medium. A novel isoenzyme, named Tvlac, was identified, purified and characterized. Its optimum pH and temperature were from 4.5 to 5 and 55 to 60 A degrees C, respectively. Its activity was stimulated by ethanol at 10 % (v/v) which increased the V (0). Conclusions The biochemical properties of Tvlac substantiate the potential of this enzyme for applications under an aqueous ethanol mixture environment.