Microfluidic paper-based analytical devices (μPADs) have revolutionized analytical chemistry by providing low-cost, portable, and efficient tools for diagnostics, environmental monitoring, and food safety. These devices rely on capillary forces for fluid transport, eliminating the need for external pumps and enabling multiplexed analysis. Fabrication techniques, such as photolithography, wax printing, and inkjet printing, enhance their versatility and adaptability. A major advancement in this field is the integration of luminescence detection techniques, including chemiluminescence and electrogenerated chemiluminescence (ECL). These methods offer high sensitivity and simplified measurements, making them highly effective for detecting hormones, drugs, and pollutants. Biospecific molecules, such as proteins and nucleic acids, enhance their selectivity. Miniaturized formats like microarrays and whole-cell biosensors further improve efficiency. Despite their strong analytical capabilities, commercialization remains challenging due to reproducibility and optimization issues. Research focuses on enhancing sensitivity, selectivity, and real-time usability while integrating portable technologies. Advances in 3D printing facilitate miniaturization and scalability, making these biosensors more practical for real-world applications. This evolving field aims to develop rapid, cost-effective, and miniaturized biosensors for medical, environmental, and food safety applications. Addressing technical and commercial challenges will be crucial for widespread adoption in research and industry.