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An understanding of the reproductive biology of aquatic organisms is crucial for the efficient conservation and management of species and/or populations. Nevertheless, conventional spawning surveys such as visual- and capture-based monitoring generally require laborious, time-consuming work and are subject to monitoring biases such as observer bias, as well as miscounts due to false spawning. In addition, direct capture often damages eggs or individuals. Thus, an efficient non-invasive approach for monitoring spawning activity on aquatic species would be a valuable tool to understand their reproductive biology and conserving biodiversity. Here, we proposed an environmental DNA (eDNA)-based approach for monitoring and understanding spawning activity by observing spikes in eDNA concentration after spawning activity. We found in cross experiments using two medaka species (Oryzias latipesandO. sakaizumii, 1:1 individual per tank) that an eDNA spike occurred in only male species after spawning activity. In addition, the magnitude of the eDNA spike was dependent on the number of spawning activities with egg and sperm release. In the field survey during the reproductive season, eDNA concentration after spawning were 3–25 times higher than before spawning. On the other hand, there was no increase in eDNA concentration during the non-reproductive season. Therefore, our results demonstrated that spikes in the eDNA concentration are mainly caused by the release of sperm during spawning activity, and it can be used as evidence of spawning in field survey. The presented approach could be a practical tool for studying reproductive biology and provides an opportunity to design effective conservation and environmental management actions.