Determining the timing and location of reproductive events for threatened species can be critical for the design and evaluation of appropriate environmental management actions. Most methods currently available for monitoring reproduction in aquatic organisms are biased, costly, time intensive and often require lethal sampling. Here, we present an environmental DNA (eDNA) based methodology, which can overcome these constraints and has the potential to accurately determine spawning time and location. During spawning, the mass release of spermatozoa, which contain few mitochondria and highly protected nuclear DNA, is a major source of eDNA. Consequently, we hypothesized that the relative abundance of mitochondrial and nuclear eDNA will change during reproductive events. Using the nationally endangered Macquarie perch, we simulated spawning in experimental tanks and monitored the changes in mitochondrial and nuclear eDNA over time. Additionally, a small scale field survey was conducted in the upper Murrumbidgee River to validate the experimental results. The results of the experimental and field based studies revealed that both target fragments are equally abundant outside of the reproductive period. In contrast, samples collected after spawning was simulated/observed contained significantly more nuclear eDNA while mitochondrial eDNA concentrations increased more moderately. Hence, the ratio between both target fragments can be used to detect recent spawning activity.