Laccases are copper polyphenol oxidases that are interesting for several applications such as in the food industry, sewage treatment and decolorization. The use of agro-industrial byproducts allows bioprocesses development for the production of large quantities at viable cost enzymes. In this study, the laccase production of Agaricus blazei was optimized in submerged cultivation (SmC). First, the following agro-industrial substrates were evaluated: sugarcane molasses, soybean molasses, coffee husks, soybean husks and pellet citrus pulp; and then these nitrogen sources: urea, ammonium sulfate, yeast extract. For the optimization of laccase production, a Plackett-Burman and 2 (4-1) incomplete factorial designs were used to evaluate the effect of KH 2 PO 4 , MgSO 4 , KCl, FeSO 4 , ZnSO 4 and the ethanol on laccase activity, and for the optimization of sugarcane molasses, urea, MgSO 4 and ethanol concentrations. Finally, laccase production kinetics was determined. The best substrate for laccase production by A. blazei was sugarcane molasses. After optimization, it was found that laccase activity of 9635 U/L was obtained in medium with sugarcane molasses (6 g/L), urea (1.5 g/L), MgSO 4 (12 mM), ethanol (1.2 mM) at 28°C and pH 8.0 during 10 days of cultivation. The results indicate that sugarcane molasses is a promising substrate for A. blazei laccase production.