The invention belongs to the related fields of biomedical products and clinical detection and the like, and mainly relates to a new method for separating and purifying wild-type p53 protein. The separation and purification method of the wild-type p53 protein comprises cloning of a p53 gene, construction of recombinant expression plasmid, protein induced expression and purification, prokaryotic system-based inducible expression, ultrasonic cracking of thalli, and separation and purification of the p53 protein in the obtained cracked supernate by using nickel column affinity chromatography. High-concentration active target protein is separated and purified by the prokaryotic system-based inducible expression and the nickel column affinity chromatography in the ultrasonic cracked thalli supernate. Denaturation and renaturation of inclusion bodies are not performed, and the protein is directly purified from the ultrasonic cracked supernate, so that the activity of the target protein is furthest kept; and compared with the conventional method, the method has the advantage that: the yield of the target protein is greatly improved by optimizing the inducible expression and using a purification scheme.