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Biosensors are analytical tools that can be used as simple, real-time, and effective devices in clinical diagnosis, food analysis, and environmental monitoring. Nanoscale functional materials possess unique properties such as a large surface-to-volume ratio, making them useful for biomedical diagnostic purposes. Nanoengineering has resulted in the increased use of nanoscale functional materials in biosensors. Various types of nanostructures i.e., 0D, 1D, 2D, and 3D, have been intensively employed to enhance biosensor selectivity, limit of detection, sensitivity, and speed of response time to display results. In particular, carbon nanotubes and nanofibers have been extensively employed in electrochemical biosensors, which have become an interdisciplinary frontier between material science and viral disease detection. This review provides an overview of the current research activities in nanofiber-based electrochemical biosensors for diagnostic purposes. The clinical applications of these nanobiosensors are also highlighted, along with a discussion of the future directions for these materials in diagnostics. The aim of this review is to stimulate a broader interest in developing nanofiber-based electrochemical biosensors and improving their applications in disease diagnosis. In this review, we summarize some of the most recent advances achieved in point of care (PoC) electrochemical biosensor applications, focusing on new materials and modifiers enabling biorecognition that have led to improved sensitivity, specificity, stability, and response time.