Response surface methodology (RSM) was applied to optimize the removal of flumequine (FLU) from aqueous solution by laccase from Trametes versicolor. Box-Behnken design (BBD) with four variables namely pH, temperature, FLU initial concentration, and 2,2-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) concentration was used to optimize these factors. The results showed that the predicted values for FLU removal were close to the experimental values, and the R 2 (0.9967) indicated that the regression was able to give a good prediction of response for the FLU removal process in the studied range. Optimization of the factors levels was carried out in two approaches. At first, all factors were set in the studied range. The selected optimal conditions for the maximum removal of FLU (98.27%) were predicted as: temperature of 39.64˚C, pH of 4.06, FLU initial concentration of 90.74 mg L −1 , and ABTS concentration of 1.35 mM. Then, the optimization was carried out by minimizing the amount of ABTS, maximizing the FLU initial concentration, and maximizing the removal efficiency. The recommended optimum conditions are: temperature of 39.19˚C, pH value of 4.32, FLU initial concentration of 125 mg L −1 , and ABTS concentration of 0.28 mM with the percentage FLU removal of 77.76%.