Abstract Reactive oxygen species (ROS) are important factors mediating aging according to the free radical theory of aging. Few studies have systematically measured ROS levels in relationship to aging, partly due to the lack of tools for detection of specific ROS in live animals. By using the H2O2-specific fluorescence probe Peroxy Orange 1, we assayed the H2O2 levels of live Caenorhabditis elegans with 41 aging-related genes being individually knocked down by RNAi. Knockdown of 14 genes extends the lifespan but increases H2O2 level or shortens the lifespan but decreases H2O2 level, contradicting the free radical theory of aging. Strikingly, a significant inverse correlation between lifespan and the normalized standard deviation of H2O2 levels was observed (p < 0.0001). Such inverse correlation was also observed in worms cultured under heat shock conditions. An oxidative fluctuation hypothesis of aging is thus proposed and suggests that the ability of animals to homeostatically maintain the ROS levels within a narrow range is more important for lifespan extension than just minimizing the ROS levels though the latter still being crucial.