Electrochemical sensing is an efficient and inexpensive method for detection of a range of chemicals of biological, clinical, and environmental interest. Carbon materials-based electrodes are commonly employed for the development of electrochemical sensors because of their low cost, biocompatibility, and facile electron transfer kinetics. Electrospun carbon fibers (ECFs), prepared by electrospinning of a polymeric precursor and subsequent thermal treatment, have emerged as promising carbon systems for biosensing applications since the electrochemical properties of these carbon fibers can be easily modified by processing conditions and post-treatment. This review addresses recent progress in the use of ECFs for sensor fabrication and analyte detection. We focus on the modification strategies of ECFs and identification of the key components that impart the bioelectroanalytical activities, and point out the future challenges that must be addressed in order to advance the fundamental understanding of the ECF electrochemistry and to realize the practical applications of ECF-based sensing devices.