The enzyme lactate dehydrogenase – LDH catalyses the final reaction of glycolysis, the interconversion of pyruvate and lactate. The determination of this enzyme in serum has received much attention because is useful in the diagnosis of diseases involving damage to tissues. The purified enzyme can be useful to biomedical analysis performing the calibration curves to measurements of the enzyme activity, kinetic and stability studies and structural analyses. The aqueous two-phase systems (ATPSs) allow enzyme separations based on molecular mass, conformation, charge and / or hydrophobicity. Aqueous two-phase systems (ATPSs) composed of polyethylene glycol (PEG) and citrate were used for partition of lactate dehydrogenase (LDH) from bovine heart crude extract. The two-level factorial design was used and in analyze of the results and a particular attention was paid to the influence of the PEG molecular mass in the purification factor of LDH. The lesser PEG molecular mass (400) in higher concentrations led to a better interaction between PEG and LDH. In the system performed by 42% (w/w) PEG 400 and 12.5% (w/w) citrate, the highest purification factor in the top phase (7.9) was obtained with an enzyme yield around 100%. However, the decrease in PEG molecular mass led to a better purification factor and yield of the LDH.