Si-based antiresonant reflecting optical waveguide type B (ARROW-B) surface plasmon resonance (SPR) biosensors allow label-free high-sensitivity detection of biomolecular interactions in real time. The ARROW-B waveguide, which has a thick guiding layer, provides efficient coupling with a single-mode fiber. The Si-based ARROW-B SPR biosensors were fabricated by using the standard semiconductor fabrication processes with a single-step lithography. A fluid flow system was designed to transport samples or analytes. The waveguide consists of propagation and SPR sensing regions. The propagation regions in the front and rear of the SPR sensing region have a symmetric cladding structure to isolate them from environmental changes. A high-index O-ring is used to seal the liquid flow channel. The intensity interrogation method was used to characterize the sensors. The sensitivity of the biosensors was 3.0 × 103 µW/RIU (refractive index unit) with a resolution of 6.2 × 10−5 RIU. An in situ regeneration process was designed to make the sensors reusable and eliminate re-alignment of the optical measurement system. The regeneration was realized using ammonia-hydrogen peroxide mixture solutions to remove molecules bound on the sensor surface, such as self-assembled 11-mercapto-1undecanoic acid and bovine serum albumin. SPR was used to monitor the regeneration processes. The experimental results show that the sensing response did not significantly change after the sensor was reused more than 10 times. In situ regenerations of the sensors were achieved.