This study sought to explore the role of the IFN-related innate immune responses (IFN-β and IFN-λ) and of reactive oxygen species (ROS) after influenza A virus (IAV) infection for antiviral innate immune activity in normal human nasal epithelial (NHNE) cells that are highly exposed to IAV. Passage-2 NHNE cells were inoculated with the IAV WSN/33 for 1, 2, and 3 days to assess the capacity of IFN and the relationship between ROS generation and IFN-λ secretion for controlling IAV infection. Viral titers and IAV mRNA levels increased after infection. In concert with viral titers, we found that the generation of IFNs, such as IFN-β, IFN-λ1, and IFN-λ2/3, was induced after IAV infection until 3 days after infection. The induction of IFN-λ gene expression and protein secretion may be predominant after IAV infection. Similarly, we observed that intracellular ROS generation increased 60 minutes after IAV infection. Viral titers and mRNA levels of IAV were significantly higher in cases with scavenging ROS, in cases with an induced IFN-λ mRNA level, or where the secreted protein concentration of IFN-λ was attenuated after the suppression of ROS generation. Both mitochondrial and dual oxidase (Doux)2-generated ROS were correlated with IAV mRNA and viral titers. The inhibition of mitochondrial ROS generation and the knockdown of Duox2 gene expression highly increased IAV viral titers and decreased IFN-λ secretion. Our findings suggest that the production of ROS may be responsible for IFN-λ secretion to control IAV infection. Both mitochondria and Duox2 are possible sources of ROS generation, which is required to initiate an innate immune response in NHNE cells.