Xylanase enzyme degrades linear polysaccharide beta-1,4 xylan and the hemicellulose of the plant cell wall. There is a growing demand in finding a cost-effective alternative for industrial scale production of xylanase with high purity for pharmaceutical applications. In this study, an alcohol/salt aqueous biphasic system (ABS) was adopted to recover xylanase from the Bacillus subtilis fermentation broth. The effects of several ABS parameters such as types and concentrations of alcohols and salts (i.e., sulphate, phosphate, and citrate), amount of crude loading and pH of the system on the recovery of xylanase were investigated. Partition coefficient of xylanase (K-E), selectivity (S) and yield (Y-T) of xylanase in top phase of the ABS were measured. Highest K-E (6.58 +/- 0.05) and selectivity (4.84 +/- 0.33) were recorded in an ABS of pH 8 composed of 26% (w/w) 1-propanol, 18% (w/w) ammonium sulphate. High Y-T of 71.88% +/- 0.15 and a purification fold (P-FT) of 5.74 +/- 0.33 were recorded with this optimum recovery of xylanase using alcohol/salt ABS. The purity of xylanase recovered was then qualitatively verified with sodium dodecyl sulphate (SDS) gel electrophoresis. The SDS profile revealed the purified xylanase was successfully obtained in the top phase of the one-step 1-propanollsulphate ABS with a distinct single band. (C) 2017, The Society for Biotechnology, Japan. All rights reserved.