Objective：To identify Alu RNA expressed in oxidative-stressed A549 cells and to construct eukaryotic expression plasmids for Alu RNA and its two kinds of loss-of-function mutants so as to study the effect on oxidative stress in cells with overexpression of Alu RNA and its mutants. Methods：Alu RNA was amplified with RT-PCR from H2O2 treated A549 cells. pcDNA3.0-Alu（pAlu），pcDNA3.0-Alu-left arm（pAlu-LA）and pcDNA3.0-Alu G25C（pAlu-M）were constructed by directional cloning and point mutation. After recombinant transfection，reactive oxygen species（ROS） produced in transfected A549 cells was detected by DCFH-DA. Results：Alu fragment was successfully amplified from stressed A549 cells. Restriction analysis and DNA sequencing proved that recombinant eukaryotic expression plasmids of pAlu，pAlu-LA and pAlu-M were constructed as expected. pAlu or pAlu-LA transfection promoted ROS production significantly in A549 cells，while pAlu-M transfection induced ROS production was markedly decreased as compared with pAlu transfection. Conclusion：The eukaryotic expression plasmids for Alu RNA and its loss-of-function mutants were successfully constructed from oxidative-stressed A549 cells. Data in vitro suggested that Alu RNA promoted ROS production in A549 cells with translational regulation.