Abstract The partitioning of glucose-6-phosphate dehydrogenase (G6PDH) (E.C. 22.214.171.124) and hexokinase (E.C. 126.96.36.199) in polyethylene glycol (PEG)–hydroxypropyl starch (PES) and PEG–phosphate aqueous two-phase systems was investigated with free triazine dyes, Cibacron Blue F3GA and Procion Red HE3B, as their affinity ligands. It was found that the free reactive triazine dyes, not bound to phase-forming polymers, preferentially partitioned in the top-PEG phase in the PEG–salt and PEG–PES systems. The effect of various parameters such as type and concentration of affinity ligands, pH of the system, molecular mass of PEG and phase composition on partitioning of the enzymes was estimated. Phosphate is a key factor affecting the enzyme partitioning in the PEG–PES system. Cibacron F3GA changed the partition coefficient of G6PDH from 0.73 to 1.59.