Four different polyethylene glycol-phosphate aqueous two-phase systems were investigated in terms of improving the efficiency of fungal laccase partitioning. Each system was composed of the same phosphate buffer (pH = 7.0) and different polyethylene glycol (PEG) (400, 1500, 3000, 6000 g/mol). Two types of laccase supernatants from cultures of Cerrena unicolor and Pleurotus sapidus were chosen for the experiments. The influence of PEG molecular weight, tie line lengths, and phase volume ratios on laccase partitioning and recoveries were examined. Additionally, the influence of individual phase forming components on the activity of the enzymes was investigated. Molecular weight of PEG had substantial effect on laccase partitioning. Influence of tie line length on activity partitioning efficiency varied significantly, depending on the type of laccase. Phase volume ratios had decisive influence on partitioning coefficients between phases. In the most successful experiments, activity partitioning coefficients tended to infinity, with recoveries reaching over 90%. Much different behavior of laccases from the two hosts was observed and discussed, as C. unicolor laccases showed much higher affinity towards the salt-rich phase than P. sapidus laccases. Molecular weights, isoelectric points, and glycosylation of laccases were investigated as possible causes for partitioning differences, of which glycosylation was the deciding factor.