Recombinant Fusarium solani pisi cutinase is generally purified by a multistep process that includes (a) osmotic shock, (b) acid precipitation, and chromatography. Steps a and b provide a combined yield of only 77% with a purification factor of 4.1. We report a single-step process using an aqueous two-phase partitioning system of 25% (w/w) polyethylene glycol 1000 and 10% (w/w) sodium phosphate, pH 4.8, to obtain 91% yield with a purification factor of 4.1. This protocol is completed within 20 min, is a cheap alternative to steps a and b, and is easy to scale up.