In recent years, natural polymer‐based electrospun fibers (EFs) with huge specific surface area, good biocompatibility, and biological activity obtained from electrospinning process exhibit tremendous vitality in the field of biomedical areas. Herein, the parameters of electrospinning from two perspectives, polymer solution such as solvent, polymeric relative molecular mass, concentration, viscosity, and conductivity of the solution, and electrospinning process such as spinning voltage, spinning flow rate, needle tip to collector distance, temperature, and humidity are first detailed. Next, the raw materials consisting of polysaccharides such as cellulose, hyaluronic acid, alginate, and chitosan as well as proteins such as collagen, gelatin, silk fibroin, and keratin are summarized. The preparation method and related characteristics of EFs with multistage structures such as porous, core–shell, Janus, bamboo‐like and other structures are introduced. The biomedical applications of these natural polymer‐based EFs mainly including tissue engineering, drug sustained release, wound dressings, and biomedical sensors are systematically recapitulated. Finally, the outlook on natural EFs is further proposed.