Ubiquitin is a small protein that widely exists in all eukaryote whose basic function is through the ubiquitin-proteasome pathway (UPP）to let the targeting protein degradation effective and highly selectivity. A cDNA fragment encoding ubiquitin-52aa extension protein from the third instar larvae of Plutella xylostella was amplified through RT-PCR and RACE method. The ubiquitin-52aa extension gene in P.xylostella, named Px-ubi, was 537 bp in total length and 387 bp in ORF. It encoded a peptide of 129 amino acid residues (GeneBank No.FJ527489), in which the relative quality of molecules is 16.7 ku and the isoelectric point is 9.1.Multiple sequence alignment indicated that Px-ubi was similar to the homologous proteins of other eukaryotic species and it shared 92% to 98% amino acid sequence identity with other species. The theoretical three dimensional structure of Px-ubi was displayed by homology modeling. Through the RT-PCR, it was found that Px-ubi was expressed at different times in the P.xylostella, and its content was higher in the egg stage, third instar larvae and pre-pupal stage than in the adult stage. The Px-ubi was then inserted into expression pET-32a (+) and transformed into E.coli DE3. Western blotting indicated that the Px-ubi was expressed successfully in the BL21 strain of Escherichia coli induced with IPTG. The results provide some helpful information for the further study of the function of ubiquitin gene in insect.