Rhipicephalus microplus is the main blooding-sucking ectoparasite of bovines and is regarded as important vectors of animal diseases such as Babesiosis. Mining protective antigens of R. microplus to develop antitick vaccine is the most potential tick control strategy. In this study, the specific primers were designed according to the conserved nucleotide sequence of enolase gene in Haemaphysalis flava, Ixodes ricinus, and Ornithodoros moubata. The fragment of enolase gene was obtained by PCR using cDNA template from fully engorged female R. microplus. The full-length cDNA of enolase gene was amplified using rapid amplification of cDNA ends (RACE). Expression pattern of enolase gene in different tissues of R. microplus was analyzed by real-time quantitative PCR (qRT-PCR). Results showed that the full-length enolase cDNA containing 2052 bp was obtained successfully. The complete cDNA included an ORF of 1305 nucleotides encoding a protein of 434 amino acids. The enolase exhibited 85.0% amino acid identity to the enolase of H. flava, 81.1% to I. ricinus enolase, and 81.3% to O. moubata enolase. qRT-PCR analysis indicated that the enolase had the highest expression in the salivary gland of R. microplus.