The operational conditions for an aqueous two-phase system (ATPS) for beta-galactosidase purification were optimized and applied to the design of a purification strategy as an alternative to the primary purification steps. The ATPS proved to be suitable for the recovery and primary enzyme purification. The purification process design developed by ATPS, diafiltration, ion exchange, and diafiltration/ultrafiltration was successful, yielding a more than tenfold purification. The purification strategy design resulted in a powerful integrated purification and recovery process, an evidence of the potential for a scale-up of the beta-galactosidase purification process.