The invention discloses ubiquitin-like substrate fusion protein as well as a preparation method and medical application, and provides a novel NEDD8 (neural precursor cell-expressed developmentally downregulated 8) modified fusion protein, which solves the technological problem of dissolvable expression of PLA2 on escherichia coli. A pColdTF cold-induced expression carrier is utilized by the preparation method to effectively prepare the fusion protein Strep tagII-NEDD8-PLA2, so the defects that the PLA2 protein expression amount is low, the expression product existing in a inclusion body form brings the inconvenience for subsequent purification in the prior art, the reactogenicity and immunogenicity of the expression product is poorer than the soluble protein and the like are overcome.