In order to clarify the sequence characterisation and localisation analysis of the ubiquitin-like protein in Enterocytozoon hepatopenaei (EhUBL), the sequence features and spatial structure of EhUBL were predicted and analysed using online websites such as Expasy, WegoLoc and Interpro, the recombinant EhUBL (rEhUBL) was obtained by cloning its coding gene EhUBL and prokaryotic expression and the specific polyclonal antibody (anti-EhUBL) was prepared, and then the subcellular localisation of EhUBL was probed by immunoelectron microscopy. The results showed that: 1) The length of EhUBL was 873 bp, it encoded 292 amino acids; the predicted relative molecular mass of EhUBL was 31.38 kDa, and the protein had the UBL (Ubiqutin-like)-UBA (Ubiquitin associated) domain. 2) EhUBL was closely related to the hypothetical protein ECANGB1_154 of Enterospora canceri and the hypothetical protein EBI_25559 of Enterocytozoon bieneusi. 3) rEhUBL was successfully obtained by gene cloning and prokaryotic expression, and anti-EhUBL was also successfully prepared. 4) EhUBL was mainly distributed in the cell wall and nucleus of the spore of E. hepatopenaei. The results above laid a foundation for the functionl study of EhUBL and provided a new target for the prevention and control of E. hepatopenaei.