Screening of effective Trametes strains with high laccase-productivity and optimization of conditions on laccase production by the diameters of colored rings by adding guaiacol to the solid medium and assaying the diameters of colored rings, we got the target strain Trametes orientalis Cui 6300. Meanwhile, the optimal conditions of strain Cui 6300 for laccase production were obtained by means of single factor analysis, orthogonal test and ABTS method: maltose 15 g/L, peptone 3 g/L, pH 4.8, Cu²⁺ 2.0 mmol/L, culture temperature 28 °C, inoculum 1.5 cm in diameter, then the highest activity was 19.923 U/mL. Besides, the influences of Cu²⁺ concentration and adding time on biomass and laccase activity were tested, and the optimal concentration and adding time were 2.0 mmol/L and on day 3 of culturing, respectively.