In this work the validation of a continuous enzyme extraction using aqueous two-phase system (ATPS) inside a tubular mixer settler with three equilibrium stages in counter-current arrangement is described. Stocks of 50% (w/w) PEG1000 and 35% (w/w) phosphates (pH = 7) were mixed to obtain a PEG1000 Phosphate batch and continuous system (TLL: 40.85%). A commercial food colorant was employed to observe phase formation inside the device. By means of mass balance and correlations of density, flow rates and phase compositions, the equilibrium behaviour of phases was converged through iterations. The phases flowing at 3.5 L/h reach steady state in no more than 30 min. Invertase extraction from spent yeast was selected as the experimental model, comparing its partitioning behaviour in batch and continuous ATPS. The distribution ratio of invertase versus impurities after three equilibrium stages (K-U = 0.470), reached the partitioning coefficient of batch system (K-U = 0.453) in 25 min. Although the total unoptimized recovery of invertase was no more than 31.4% (5.4 +/- 0.13 mU/mg), but comparable from that from the batch systems, the system proposed here, allow the use of continuous ATPS as a process stage, handling a crude extract from a complex sample. This practical approach highlights ATPS as an alternative primary recovery continuous stage in the biotechnological industry. (C) 2014 Elsevier B.V. All rights reserved.