Abstract: 4-coumarate: coenzyme A ligase is a key enzyme in lignin synthesis pathway. A 4CL gene was cloned from the young stem of Eucalyptus urophylla clone GLU4 and named Eu4CL using specific primers based on the highly conserved sequences of plant 4CL. The gDNA and cDNA sequence were 5 534 bp and 1 640 bp respectively, while CDS encoded 544 amino acid residues. Eu4CL had 98% sequence homology with Eucalyptus that had been logged in GenBank, and its homology with Phyllostachys and others were over 77%. Eu4CL encoding sequence was analyzed, the result showed it has an entire 4CL domain, an AMP binding site and a CoA binding site, the homology with Cinnamomum osmophloeum and others were over 79%. The physicochemical property, structure of Eu4CL and its phylogenetic analysis were analyzed using bioinformatics tools and MEGA. The SDS-PAGE analysis showed that Eu4CL was transformed into pQE30/M15 system and fusion protein with molecular weight about 60 ku was successfully expressed in transformant. The cloning and prokaryotic expression of Eu4CL gene provided effective resources for enzymology research and futher research of transgenic.