Extraction of lipoxygenase-1 was investigated by using the aqueous two-phase system formed by sodium sulfate−poly(ethylene glycol)−buffer. The extraction was performed in a 47-mm inner diameter spray column, operating in a semibatch manner. The effects of dispersed-phase flow rate and phase compositions on fractional dispersed-phase hold up (ε_D) and volumetric mass-transfer coefficient (K_Da) were respectively studied. It was found that the hold-up and volumetric mass-transfer coefficient increased with an increase in dispersed-phase velocity and decreased with increasing phase composition. Correlations have been developed by nonlinear regression for estimation of fractional dispersed-phase hold up and volumetric mass-transfer coefficients.