This study explores the production and characterization of an extracellular β-fructofuranosidase (FFase-I) by Aspergillus versicolor newly isolated from Atlantic Forest-Brazil. The β-fructofuranosidase production by fungus, after the optimization process using central composite design and response surface methodology, showed that 3% (w/v) apple pomace, an initial pH 7.5, and 12 days of cultivation provided the best conditions. The β-fructofuranosidase (FFase-I) was purified from the crude extract by 75% (NH 4 ) 2 SO 4 precipitation followed by DEAE-Sephadex, and the molecular mass of the FFase-I was estimated to be 75 kDa by SDS-PAGE. Furthermore, the enzyme exhibited unusual tolerance to Cu 2+ , sodium dodecyl sulfate (SDS), Tween 80, ethanol and acetone. The purified enzyme had an optimum pH of 6.0 and was stable over an acidic pH range of 3.0-6.0. The optimum temperature of the FFase-I was 55°C but was stable at 60°C for 7 h. Thus, the β-fructofuranosidase A. versicolor which has thermal stability and activity under acidic conditions would have potential application in sugar cane molasses hydrolysis for subsequent ethanol production.