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S-layer protein (SLP) is a surface protein present in many bacteria and shows unique self-assemble ability for various applications, but its detection methods are very limited. In this paper, a label-free, highly selective and sensitive assay of SLP was developed by using the TiO₂-coated porous silicon (PSi)-based microfluidic biosensor. The PSi-based biosensor could be easily prepared in a microfluidic device to build a microfluidic biosensing system for measuring SLP in samples rapidly. As the detection principle of the biosensor was based on the light reflection interference, so there was no labeling requirement. The specific recognition capability of the PSi-based biosensor was achieved by the non-covalently immobilized anti-SLP on the PSi surface. The developed biosensing system showed a rapid response time (5 min) and an excellent specificity to SLP, and displayed a broad detection linear range of 0.001 …