The phenomenon of conflicting gene trees has become a remarkable and difficult problem. Application of multiple genes has been a widespread practice to reconstruct phylogenies in phylogenetic studies. Enolase is a key glycolytic enzyme, The enzymes from a large variety of organisms, including archaebacteria, eubacteria and eukaryotes, were studied. We downloaded eno sequences from the genomes of bacteria and archaea that have been completely sequenced. The comprehensive homology search and phylogenetic analysis of the eno were used, and nineteen horizontally transferred genes were identified. The results of analysis showed lots of differences between the features of horizontal transferred genes and the ones of whole genomic genes, such as nucleotide composition, gene combination, codon usage bias, and selection pressure. These results reconfirmed that the horizontally transferred genes were exogenous. The result revealed that prokaryote eno genes were highly conserved, medium-sized, is a good material in the phylogenetic. This paper can provide a reference in study of life habit and evolutionary history of donor and receptor, and enolase structure and function.