Objective: To construct eukaryotic expression vector of adenovirus type 5 fiber gene and investigate its expression in eukaryotic cell. Methods: By endonuclease digestion, fragment harboring the fiber gene was cloned into plasmid pBluescript M13- from the pAdEasy-1 to construct pBS/Fiber. The pBS/Fiber was then digested with XbaI and KpnI. The yielding fragment was subcloned into the pcDNA3.1, obtaining the eukaryotic expression vector pcDNA/Fiber. To test the eukaryotic expression ability of the pcDNA/Fiber, pcDNA/Fiber was transiently transduced into the COS-7 cells using lipofectamine, and the fiber protein was detected by SDS-PAGE and Western blot. Results: Both plasmid pcDNA3 1 and eukaryotic expression vector pcDNA/Fiber were successfully constructed. Anew protein band was detected by SDS-PAGE and Western blot, its molecular weight was 62 kD on denaturing SDS-PAGE gel and 186 kD on non-denaturing SDS-PAGE gel.Conclusions: The eukaryotic expression vector of the adenovirus type 5 fiber gene was expressed in the COS-7 cells. In natural state, the expressed fiber protein was a trimer. The plasmid pBS/Fiber can be used to construct target adenovirus vector.