Synthetic biology-based biosensors represent a next-generation combination of biological and engineering systems that offer a versatile nature, and they can be easily modified, making them sensitive tools for the detection of analyte molecules in various fields. By using engineered microorganisms, advanced genetic circuits, and cell-free expression systems that include CRISPR-Cas technology, these biosensors provide several advantages with respect to programmability, adaptability, and specificity as compared to conventional biosensors. Their modular framework consists of advanced signal identification, processing, and response modules that allow immediate and quantifiable translation of biological molecules into measurable signals. This chapter highlights the major progress in the application of biosensors, ranging from point-of-care diagnostics, real-time monitoring of diseases, and personalized healthcare to the detection of pollutants that harm the environment and monitoring in the fermentation industry. Artificial intelligence (AI) has led to portable and smart devices, which offer real-time diagnostic platforms that are capable of detecting multiple analytes. Despite their promise, challenges such as signal reliability, adaptation to complex sample workflows, and long-term performance are the active areas in which research is ongoing. The combination of synthetic biology, AI, nanotechnology, and microfluidics leads to the innovation of better methods, highlighting synthetic biosensors as an innovative analytical platform that is capable of transforming diagnostics, monitoring, and management in healthcare, the environment, and industrial applications.