Ultrasensitive and specific detection of low-abundance tumor biomarkers remains a major challenge for early and minimally invasive cancer diagnosis. Here, we present a high-performance biosensing platform that integrates a genetically engineered bacterial S-layer with an AlGaN/GaN high-electron-mobility transistor (HEMT) sensor for label-free detection of tumor antigens. As a proof-of-concept, the ovarian cancer antigen human epididymis protein 4 (HE4) was selected . Specifically, the S-layer protein rSbpA was fused with HE4-specific nanobody 1G8 to construct a bifunctional membrane capable of self-assembling into an ordered biorecognition layer on the sensor surface. Compared to conventional chemical crosslinking, S-layer-driven assembly increased antibody loading by 50 % and minimized nonspecific adsorption in plasma environments. The resulting HEMT sensor detected HE4 across a dynamic linear range (10⁻²¹ to 10⁻¹⁴ M), identifying patients with ovarian cancer with 100 % diagnostic accuracy (AUC = 1.0). This study establishes a versatile and modular biosensing strategy for ultra-low-abundance biomarker detection with broad potential applications in the precision diagnostics of cancer and other diseases. ... Read more