Electrochemical sensors have become indispensable tools for the detection and monitoring of food and environmental toxins. In recent years, nanofiber-based materials have emerged as promising candidates for constructing electrochemical sensors, offering a plethora of unique advantages, such as a large surface area, enhanced electron transfer kinetics, and the ability to immobilize diverse recognition elements. This comprehensive review sheds light on the pivotal role of nanofibers in advancing electrochemical sensors for the detection of food and environmental toxins. Various fabrication methods for nanofibers, including electrospinning, template-assisted synthesis, and self-assembly techniques, are elucidated in detail. It highlights the integration of nanofibers as electrode materials, nanocomposites, and immobilization platforms for recognition elements like enzymes, antibodies, and aptamers. The influence of nanofiber properties, encompassing morphology, composition, and surface modification, on the sensing performance is meticulously discussed. Moreover, a comprehensive overview of the latest advancements in nanofiber-based electrochemical sensors for detecting pesticide residues, heavy metals, mycotoxins, and other environmental contaminants is presented. We address challenges and future perspectives in the field, including scalability, cost-effectiveness, and seamless integration with wearable devices. This review illuminates new horizons for the development of sensitive, selective, and portable analytical devices, thereby significantly contributing to improved food safety and environmental monitoring.