The invention discloses recombinant spider silk protein, a coding gene thereof, an expression and purification method and application of the recombinant spider silk protein in the fields of silk formation, film formation and the like. The recombinant spider silk protein consists of an N-terminal non-repeated area (N-NRT), a core repeated area (REP), and a C-terminal non-repeated area (C-NRT), wherein the core repeated area consists of 1-3 ADF3Reps and / or 1-6 REPAs. A series of novel spider silk protein sequences designed by using the recombinant spider silk protein integrate the main characteristics, such as high strength and high elasticity, of spider silk. Moreover, the sequences are introduced into escherichia coli through a genetic engineering technology separately, a corresponding high-density fermentation process is established, the high-efficiency prokaryotic expression of the spider silk protein is achieved, and the fermentation yield can reach 0.5 g / L or above. In addition, aninventor develops a non-chromatographic protein purification method according to the properties of the spider silk protein, and the purity of the purified recombinant spider silk protein meets the requirements of silk formation or film formation.