Determining the time and location of reproduction for threatened species is critical to design and evaluate management actions. The current available methods for monitoring reproduction in aquatic species are often biased, costly, time intensive and sometimes require lethal sampling. Here we present an environmental DNA (eDNA) based methodology for monitoring spawning activity, which can overcome these constraints. During spawning the mass release of spermatozoa, which contain few mitochondria and highly protected nuclear DNA, forms a major source of eDNA. Thus, we hypothesized that the relative abundance of mitochondrial and nuclear eDNA will change during reproductive events. Through an experimental and field-based study, focusing on the endangered Macquarie perch, we were able to show that both target fragments are equally abundant outside of the reproductive period. However, after the release of spermatozoa a strong increase in nuclear eDNA was observed while mitochondrial eDNA concentration only increased moderately. Hence, the changes in the ratio between nuclear and mitochondrial eDNA can be indicative of recent spawning activity and can be used to monitor reproductive activity in species relying on external fertilization.