Abstract: EB1, as a microtubule plus end binding protein, plays an important role in the microtubule polymerization and microtubules equipment found on the protein complex. In current experiment, EB1 gene was cloned on the basis of half smooth tongue sole cDNA library. The gene contained an open reading frame of 777 bp code that encoded putative 258 amino acids with a calculated molecular mass of 29. 497 kDa. The deduced amino acid sequence was aligned with other species, and they had the high identity and homology. It was found that the proteins included two conservative domain structure, CH and EB1, and two conservative trigeminy amino acid sites, FYF and EEF/Y. Homologous clustering analysis results showed that the EB1 proteins could well reflect the species development of evolution from lower to higher states. Real-time fluorescent quantitative PCR results showed that the genes expressed in all the tissues selected and, the highest level existed in ovary, which was higher 14.6 times than the lowest amount of liver tissue. Consistent with qRT-PCR results, cell mobility showed consistency with the expression of EB1 in tissues.