Eugenol synthase was one of the key enzymes in the synthesis pathway of methyleugenol, which was the main active constituent in Asarum heterotropoides. Gene clone and functional analysis of AhEGS would lay the foundation for revealing the synthesis pathway of methyleugenol and its metabolism regulation mechanism. Specific primers were designed according to the sequence screened in the A. heterotropoides transcriptome database, the ORF sequence of AhEGS was amplified by RTPCR, and the corresponding bioinformatics analysis was performed. Realtime fluorescent quantitative PCR method was used to analyze the expression of AhEGS in different developmental stages (young leaf period, flower period, fruit period) and different tissue parts (leaf, rhizome, root). Also we performed prokaryotic expression analysis. The results indicated that: (1) the ORF sequence of the AhEGS gene was 951 bp in length, encoding 316 amino acids, with a theoretical molecular weight of 34.93 kD and an isoelectric point of 6.19. It was a hydrophilic protein with no transmembrane structure and no signal peptide sequence. AhEGS had the highest homology with rose RcEGS (AFQ98278.1). (2) Realtime quantitative PCR analysis showed that the expression of AhEGS was highest in roots at young leaf stage. (3) The prokaryotic expression vector pET28aAhEGS BL21 was successfully constructed and transformated into Escherichia coli(E.coli) BL21(DE3). It was showed that about 35 kD of specific proteins was induced in E.coli by SDSPAGE, which was consistent with the theoretical molecular weight; the best induction condition was 16 ℃, 14 h, and 0.2 mmol/L IPTG. It was the first time to clone AhEGS gene. In this study, the prokaryotic expression vector for AhEGS was constructed and also the optimal induction conditions were screened out. These results might provide scientific evidence for the application of methyleugenol metabolic engineering.