Xylose reductase enzyme (E:C 1.1.11.21) produced by Candida guilliermondii in sugarcane bagasse was extracted by reversed micelles of N-benzy-N-dodecyl-N-bis (2-hydroxyethyl) ammonium chloride cationic surfactant. An experimental design was employed to evaluate the influences of the following factors on the enzyme extraction: temperature, cosolvent, and surfactant concentration. A model was used to represent the enzyme recovery and fit of the experimental data. The extraction yielded a total recovery of 130%, and the purity increased 4.8-fold. This study demonstrates that liquid-liquid extraction by reversed micelles is a process able to recover and increase the enzymatic activity and purity of XR produced by C. guilliermondii.