Electrospun nanofibers (NFs) have shown excellent properties including high porosity, abundant active sites, controllable diameter, uniform and designable structure, high mechanical strength, and superior resistance to external destruction, which are ideal nanoreactors for in situ characterizations. Among various techniques, in situ transmission electron microscopy (TEM) has enabled operando observation at the atomic level due to its high temporal and spatial resolution combined with excellent sensitivity, which is of great importance for rational materials design and performance improvement. In this review, the basic knowledge of in situ TEM techniques and the advantages of electrospun nanoreactors for in situ TEM characterization are first introduced. The recent development in electrospun nanoreactors for studying the physical properties, structural evolution, phase transition, and formation mechanisms of materials using in situ TEM is then summarized. The electrochemical behaviors of carbon nanofibers (CNFs), metal/metal oxide NFs, and solid‐electrolyte interphase for different rechargeable batteries are highlighted. Finally, challenges faced by electrospun nanoreactors for in situ TEM characterization are discussed and potential solutions are proposed to advance this field.image