The present invention belongs to the field of biotechnology and pharmacy, and provides a fusion protein containing a fibronectin type III domain and preparation and application thereof. The structure of the fusion protein comprises a fibronectin type III domain and an insertion sequence, which can maintain the spatial conformation of an inserted protein or an active peptide, and can effectively protect an N-terminus and/or a C-terminus of a target protein, so that the target protein is not easily affected by enzymolysis, thus obtaining a longer half-life in vivo.