We used a novel approach to purify Pleurotus ostreatus laccase using the three phase partitioning (TPP) methodology. The aim of our research was to study the effect of TPP parameters on laccase purity and yield. The response surface methodology (RSM) has been applied to design the experiments to evaluate the interactive effects of the three most important operating variables: ammonium sulphate saturation (w/v) (20-80%), ratio of crude extract to t-butanol (v/v) (1:1 to 1:3), and temperature (20-60 degrees C). Using this methodology, the optimum values for the critical components were obtained as follows: ammonium sulphate saturation (w/v), 50-60%; ratio of crude extract to t-butanol (v/v) 1.0:.8; temperature 42-45 degrees C, respectively. Under optimal conditions, the experimental laccase yield and purity was 184% and 7.22-fold, respectively. SDS-PAGE and RP-HPLC revealed that the laccase was purified by TPP. The determination coefficients (R-2) were 0.9891 and 0.9728 for laccase purity and yield, respectively, indicating an adequate degree of reliability in the model. To our knowledge, the present work demonstrates for the first time the s uccessful application of RSM to TPP.