Objective：To construct a eukaryotic expression plasmid carrying let-7a and to establish the Ewing’s sarcoma（ES） cell line A673 and SK-ES-1 stably expressing let-7a. Methods：Fragment of attB1-K- eGFP-let-7a-1-attB2 coding let-7a was amplified using Gateway protocols. The let-7a gene was recombined with donor vector pDONR221 to construct an entry plasmid pDONR221-eGFP-let-7a，which forwarded to recombine with pLV.Des2d.P/neo to reconstruct pLV.Des2d.P/neo-EF1A>eGFP/Let-7a. PCR and se－quencing were used to confirm the plasmid. Lenti-let-7a transfected ES cell lines A673/let-7a and SK-ES-1/let-7a which can sta－bly express let-7a were screened out. A673/let-7a and SK-ES-1/let-7a were confirmed by real-time PCR. Western blot analysis was performed to detect the expression of CDK6 in A673/let-7a and SK-ES-1/let-7a cells，which was the target gene of let-7a. Results：PCR results confirmed that the value of target fragment was consistent with the theoretic value. Sequencing analysis showed that let-7a was correctly inserted into the pLV.Des2d.P/neo-EF1A>eGFP/let-7a plasmid. Real-time PCR indicated that the expression of let-7a was increased remarkably in A673/let-7a and SK-ES-1/let-7a compared with that of control and blank cells；expressing values of let-7a were as high as 8.5 folds（P=0.000） and 6.5 folds（P=0.001） in A673/let-7a and SK-ES-1/let-7a respectively. Western blot analysis showed that ectopic expression of let-7a down-regulated the expression of CDK6. Conclusions：ES cell lines A673/let-7a and SK-ES-1/let-7a which can stably express let-7a are successfully established.