It was shown that by using free reactive dyes as affinity ligands, lactate dehydrogenase (LDH) can be purified with affinity partitioning directly from rabbit muscle homogenization. The free reactive dyes not bound to polyethylene glycol (PEG) showed a strong tendency toward the top PEG-rich phase in aqueous two-phase systems, and thus enhanced the affinity partitioning effect. Wide-ranged reactive dyes were screened in terms of their partitioning abilities for LDH. The effects of various parameters on affinity partitioning behavior of LDH, such as phase composition, impurities in raw material, concentration of dyes, pH of the systems, and addition of salts, were studied. The optimized affinity extraction process has been carried out for the large-scale purification of LDH from rabbit muscle homogenization. The enzyme was recovered with a yield of 81.3% and a purification factor of 7.4. Both PEG and dyes were recovered and reused directly without lowering the quality of the product.