Objective To establish a construction method for luciferase-microRNA (miRNA) sponge adenovirus, and to verify the binding efficiency of the expression product of the adenovirus and miRNA. Methods DNA fragments that can be partly complementary to miRNA-126 were chemically synthesized. The miRNA-126 sponge fragments were amplified by PCR, and cloned into pMD-18T vector and the 3'-end non-coding region of psiCheck2 plasmid; hRluc-miRNA-126 sponge was further cloned into Ad-Track plasmid. After linearization by Pme Ⅰ, Ad-Track-hRluc-miRNA-126 sponge plasmid was transformed into competent Escherichia coli strains BJ5183 to homologous recombinate with Ad-Easy plasmid; recombinant Ad-Easy-hRluc-miRNA-126 sponge plasmid was identified by Pac Ⅰ, and then transfected into 293 cells to produce adenovirus. Adenovirus was used to infect human umbilical vein endothelial cells (HUVECs), and the effect of miRNA-126 on cell migration ability was detected by scratch test. Results The miRNA-126 sponge had a good complementary base-pairing relationship with miRNA-126; double luciferase experiment showed that miRNA-126 could directly act on hRluc-miRNA-126 sponge. Cloning hRluc-miRNA-126 sponge into Ad-Track plasmid made it possible to synchronously monitor the efficiency of cellular transfection and miRNA inhibition. Ad-Easy-hRluc-miRNA-126 sponge plasmid was successfully established by homologous recombination of Ad-Track and Ad-Easy, identified by Pac Ⅰ digestion with 4.5 kb fragments. When transfected into 293 cells, luciferase-miRNA sponge adenovirus was obtained 7 days later. Overexpression of miRNA-126 inhibited the expression of hRluc-miRNA-126 sponge (P<0.01). Infection with miRNA-126 sponge adenovirus inhibited the migration of HUVECs (P<0.05). Conclusion Luciferase-miRNA sponge adenovirus has been successfully established. MiRNA-126 has a high binding efficiency with the expressed product of the adenovirus.