A novel laccase (Tolacc-T) from white rot fungus Trametes orientalis was enriched to apparent homogeneity with a specific activity of 20.667U/mg protein and recovery yield of 47.33%. The SDS-PAGE gave a single band indicating that Tolacc-T appears as a monomeric protein with a molecular mass of 44.0kDa. Domain structure analysis revealed that Tolacc-T contained a typical copper II binding domain and shared three potential N-glycosylation sites, but had no copper I binding domain, demonstrating that the enzyme is really a laccase, but a novel laccase. Optimal pH and temperature of Tolacc-T was 4.0 and 80°C, respectively, and it retained more than 80% of its original activity after 2h incubation at 10°C to 50°C. The enzyme exhibited strict substrate specificity towards ABTS but showed no or trace activities towards other substrates. Among the metals tested, Mn was proved to be the best activator for enhancing the laccase activity. A strongly inhibiting effect was found when NaN, -cysteine, and DTT were added to the enzyme. However, Tolacc-T activity was little bit inhibited in the presence of chelator EDTA. Furthermore, the enzyme was capable of degrading structurally different synthetic dyes in the absence of a redox mediator.