The growing threat of organic pollution to surface waters necessitates the development of rapid and scalable monitoring tools that transcend the limitations of the standard 5-day biochemical oxygen demand (BOD₅) test. This study presents a novel approach by developing a highly stable and rapid BOD biosensor based on the microorganism Paracoccus yeei, immobilized within a sol–gel-derived xerogel matrix synthesized on a polyhydroxybutyrate (PHB) substrate. The PHB-supported xerogel significantly enhanced microbial viability and sensor stability. This biosensor demonstrated a correlation (R² = 0.93) with the standard BOD₅ method across 53 diverse water samples from the Tula region, Russia, providing precise results in just 5 min. The second pillar of our methodology involved analyzing multi-year Landsat satellite imagery via the Global Surface Water Explorer to map hydrological changes and identify zones of potential anthropogenic impact. The synergy of rapid ground-truth biosensor measurements and remote sensing analysis enabled a comprehensive spatial assessment of water quality, successfully identifying and ranking pollution sources, with wastewater discharges and agro-industrial facilities constituting the most significant factors. This work underscores the high potential of PHB–xerogel composites as efficient immobilization matrices and establishes a powerful, scalable framework for regional environmental monitoring by integrating advanced biosensor technology with satellite observation.