Culture supernatant containing laccase produced by Cerrena unicolor strain was used to examine laccase partitioning between phases in an aqueous two-phase system. The investigated system consisted of polyethylene glycol 3000 and sodium phosphate buffer adjusted to pH = 7. Influence of several parameters on partitioning was measured, including phase forming components’ concentrations, tie line lengths, phase volume ratio, supernatant dilution, process temperature and halogen salt supplementation. Partitioning coefficients up to 78 in the bottom phase were achieved with yields of over 90%. Tie line length and phase volume ratio had significant effect on enzyme partitioning.