Laccase-catalyzed oxidation was applied in the biotransformation of levofloxacin (a potentially environmental antibiotic contamination); however, the enzyme may denature in urea-containing wastewater and lead to the formation of an inactive form followed by decreasing the yield of the bio-removal. In this study, the osmolytes-stabilized laccase was used to eliminate levofloxacin in the urea-containing solution. Sorbitol and proline 100 mM appeared to be the two most efficient laccase protectants against the urea-induced denaturation. In a 1-M urea solution, the maximum velocity (V) of laccase was estimated to be 39.1 μmol min mg. This value was improved to 101.7 and 51.8 μmol min mg in the presence of sorbitol and proline, respectively. In optimal conditions for the elimination of levofloxacin, sorbitol- and proline-treated laccase led to 82.9 % and 76.2 % bio-removal of the applied fluoroquinolone in 1 M urea solution, respectively. Biotransformation products of the parent antibiotic were spectroscopically analyzed that assigned to different reaction pathways including demethylation, defluorination, decarboxylation, deamination, and hydroxylation. A micro-toxicity study concerning the growth of some Gram and Gram bacteria exhibited decreasing in inhibition of laccase-treated levofloxacin after a 10-h incubation at 37 °C.