The invention provides a substrate for analyzing a de-Ufm1 (Ubiquitin-fold modifier 1) enzyme and a preparation method and further provides medical application of fused protein. A Ufm1protein moleculeof chickens is fused to an N end of PLA2 by utilizing the properties that the activity of the PLA2 is improved and a series of pathological and physiological processes are mediated, and a phenomenonof influencing the activity when the N end of the PLA2 is separated is very obvious; after hydrolysis reaction of a deubiquitinating enzyme to be detected is carried out, steric hindrance of the N endof the PLA2 is removed and the catalytic activity is recovered; artificial fat substrates are hydrolyzed, so that the activity of the deubiquitinating enzymes UFSP1 and UFSP2 can be directly analyzedby the substrate. The substrate provided by the invention is novel fused protein and can be used for a detection substrate of the de-Ufm1 (Ubiquitin-fold modifier 1) enzyme.