Bisulfite, a major form of SO2 in aqueous phase of apoplast, may reduce photosynthesis rate and thereby crop yield through inducing reactive oxygen species (ROS). In this study, ROS production was directly detected in a living cell of leaf of spinach (Spinacia oleracea L.) using laser scanning confocal microscopes with the assistance of the fluorescence probe dichlorofluorescin diacetate (H2DCF-DA). Results showed that, under bisulfite stress, a large quantity of ROS indicated by DCF fluorescence was produced in epidermic tissue. The role of plasma membrane (PM) NADPH oxidase in bisulfite-induced ROS production was also investigated. Treatment with bisulfite resulted in a significant increase in the content of ROS and the activity of PM NADPH oxidase in spinach leaves. The effects caused by bisulfite were inhibited pronouncedly by pretreatment with two widely used NADPH oxidase inhibitors (diphenyleneiodonium and quinacrine). Moreover, the change patterns of the bisulfite-induced increase and inhibitor-caused decrease in the two parameters were quite similar. Additionally, only a small amount of ROS could be observed on in vitro chloroplasts under bisulfite stress. Based on all the results, we conclude that ROS is involved in bisulfite-induced stress, and the bisulfite-induced enhancements in levels of ROS originate mainly from PM NADPH oxidase.