The analysis on codon usage bias of UL24 gene of duck enteritis virus (DEV) may improve our understanding of the evolution and pathogenesis of DEV and provide a basis for understanding the relevant mechanism for biased usage of synonymous codons and for selecting appropriate expression systems to improve the expression of target genes. The codon usage bias of UL24 genes of DEV and 27 reference herpesviruses were analyzed. The results showed that codon of UL24 gene of DEV was strong bias toward the synonymous codons with A and T at the third codon position. A high level of diversity in codon usage bias existed, and the effective number of codons used in a gene plot revealed that the genetic heterogeneity in UL24 gene of herpesviruses was constrained by the G + C content. The phylogentic analysis suggested that DEV was evolutionarily closer to Alphaherpesvirinae and that there was no significant deviation in codon usage in different virus strains. There were 20 codons showing distinct usage differences between DEV and Escherichia coli, 23 between DEV and Homo sapiens, but only 16 codons between DEV and yeast. Therefore the yeast expression system may be more suitable for the expression of DEV genes.