Aim: S-layer proteins are considered as a good nanocarrier due to their binding and self-assembled properties. These can be used to prepare the immunomatrixes for the removal of toxins from the samples. Methods and Results: Two S-layer proteins 70 and 40kDa of thermophilic Thermobifida fusca were extracted with guanidine hydrochloride and purified. Antibodies against S-layer proteins were developed, and their monospecificity was checked. Immunogold labelling indicated that these are surface proteins. Immunomatrixes (70-SLIM, 40 SLIM) were prepared by covalently immobilizing S-layer proteins in microwell and further conjugated with anti- Staphylococcus aureus enterotoxin B (SEB) antibodies. The binding of 70 and 40kDa proteins was observed nearly 7.0 mu g cm(-1) to binding area, and the conjugation with anti-SEB antibodies was found 1.22 mu g mu g(-1) of 70kDa and 0.875 mu g mu g(-1) of 40kDa. The average binding and elution of pure SEB toxin on 70-SLIM and 40-SLIM was 5.0 mu g SEB toxin. The SEB toxin in milk samples was also removed on immunomatrixes successfully. Conclusion: It is the first report, and this study shows that the thermophilic S-layer proteins can be used to prepare the immunomatrixes. Significance and Impact of Study: Information in this study can be used to design the strategies for the removal of biologically important materials or toxins from samples.