Point-of-care (POC) diagnostics have emerged as a critical tool in modern healthcare and agriculture, offering the potential to revolutionize disease detection and management, particularly in resource-limited settings. By enabling rapid, on-site testing without the need for specialized laboratory equipment, POC diagnostics can significantly improve patient outcomes, reduce healthcare costs, and enhance disease surveillance capabilities. Polydiacetylenes (PDAs) are a unique class of polymers with conjugated backbones that undergo a colorimetric blue-to-red transition in response to various stimuli, making them promising candidates for simple, visual detection systems. This dissertation explores the development and optimization of PDA-based sensors for point-of-use diagnostic applications. A systematic investigation of liposome synthesis parameters is conducted, identifying optimal conditions for uniform and responsive PDA assemblies. The effects of diacetylene monomer structure and lipid doping on sensor performance are examined, revealing that shorter alkyl chains and moderate lipid incorporation enhance sensitivity while maintaining stability. Efforts to develop antibody-functionalized PDA sensors for detecting plant pathogens and model proteins are described, highlighting both the potential and limitations of this approach. To overcome challenges encountered with antibody-based systems, an alternative strategy using small-molecule ligands for protein detection is explored. Throughout the work, emphasis is placed on creating robust, reproducible protocols suitable for point-of-use applications. The insights gained from this research contribute to the broader understanding of PDA-based biosensors and provide a foundation for their future development as accessible diagnostic tools for healthcare and agricultural applications.