Green electrospinning harnesses the potential of renewable biomaterials to craft biodegradable nanofiber structures, expanding their utility across a spectrum of applications. In this comprehensive review, we summarize the production, characterization and application of electrospun cellulose, collagen, gelatin and other biopolymer nanofibers in tissue engineering, drug delivery, biosensing, environmental remediation, agriculture and synthetic biology. These applications span diverse fields, including tissue engineering, drug delivery, biosensing, environmental remediation, agriculture, and synthetic biology. In the realm of tissue engineering, nanofibers emerge as key players, adept at mimicking the intricacies of the extracellular matrix. These fibers serve as scaffolds and vascular grafts, showcasing their potential to regenerate and repair tissues. Moreover, they facilitate controlled drug and gene delivery, ensuring sustained therapeutic levels essential for optimized wound healing and cancer treatment. Biosensing platforms, another prominent arena, leverage nanofibers by immobilizing enzymes and antibodies onto their surfaces. This enables precise glucose monitoring, pathogen detection, and immunodiagnostics. In the environmental sector, these fibers prove invaluable, purifying water through efficient adsorption and filtration, while also serving as potent air filtration agents against pollutants and pathogens. Agricultural applications see the deployment of nanofibers in controlled release fertilizers and pesticides, enhancing crop management, and extending antimicrobial food packaging coatings to prolong shelf life. In the realm of synthetic biology, these fibers play a pivotal role by encapsulating cells and facilitating bacteria-mediated prodrug activation strategies. Across this multifaceted landscape, nanofibers offer tunable topographies and surface functionalities that tightly regulate cellular behavior and molecular interactions. Importantly, their biodegradable nature aligns with sustainability goals, positioning them as promising alternatives to synthetic polymer-based technologies. As research and development continue to refine and expand the capabilities of green electrospun nanofibers, their versatility promises to advance numerous applications in the realms of biomedicine and biotechnology, contributing to a more sustainable and environmentally conscious future.