Objective To evaluate whether the surface layer (S-layer) protein of Lactobacillus brevis serves as a self-aggregating protein tag for cost-effective separation of human and yeast d-amino acid oxidases (hDAAO and yDAAO) expressed in E. coli. Results In aqueous two-phase (PEG-phosphate) system, the S-layer:DAAO fusion proteins (shDAAO and syDAAO) were separated at the interface with a recovery of 82 +/- 10.6% for shDAAO and 95 +/- 1.9% for syDAAO. Some shDAAO proteins were separated as precipitates with a recovery of 41 +/- 0.5% in phosphate (9%, w/w) using PEG 3000 and PEG 4000 (16%, w/w), while some syDAAO proteins were also isolated as precipitates with a recovery of 75 +/- 17.5% in phosphate (9%, w/w) using PEG 4000 and PEG 8000 (16%, w/w). Conclusions The S-layer of L. brevis was applied to a self-assembled protein tag to enable cost-effective separation of human and yeast d-amino acid oxidases expressed in E. coli cells. Because of the self-assembling properties of S-layer proteins, human and yeast d-amino acid oxidases fused with S-layer proteins could be easily separated by aggregates at the interface and/or in a few conditions by precipitates to the bottom of the PEG-phosphate aqueous system.