Triboelectric nanogenerator (TENG) is a burgeoning energy conversion technology, serving as a renewable distributed power source in the context of Internet of Things and low-carbon economy. Nanofiber mats feature light weight, porous structure, and large specific surface area, which are favourable for the performance improvement and application expansion of TENGs. As a promising technique to uniformly prepare nanofiber mats in a tailorable manner, electrospinning has attracted considerable interest since its advent. Therefore, the electrospun fiber-based TENG (EF-TENG) has evolved into a key branch in the TENG field with numerous significant research efforts. This review aims to offer a comprehensive discussion, from micro/nano scale to macro scale, on the optimization of EF-TENGs via diversified electrospinning processes and designs. Then customized applications represented by air filters, acoustic devices, electronic skins, and electronic textiles are systematically summarized. At last, the challenges and perspectives of EF-TENGs are listed in detail, providing insights for their future study and development.