To assist in detection of offshore spawning activities of the Japanese eel Anguilla japonica and facilitate interpretation of results of environmental DNA (eDNA) analysis in their spawning area, we examined the eDNA concentration released by each life history stage of artificially reared Japanese eels in the laboratory using quantitative real-time PCR (qPCR). We also compared eDNA concentrations between before and after artificially induced spawning activities. eDNA was not detected from three 30 L seawater tanks containing each single fertilized egg, but eDNA was found from other tanks each containing single individuals of larval stages (preleptocephalus and leptocephalus), juvenile stages (glass eel, elver and yellow eel) or adult stage (silver eel). The eDNA concentrations increased in the life history stages, showed a significant difference among all stages, and were positively correlated with the total length and wet weight. Moreover, the eDNA concentration after spawning was 10–200 times higher than that before spawning, which indicated that the spawning events in the ocean would produce relatively high eDNA concentration. These results in the laboratory suggested that eDNA analysis appears to be an effective method for assisting oceanic surveys to estimate the presence and spawning events of the Japanese eel in the spawning area.