Scientists increasingly rely on protected areas to assist in biodiversity conservation, yet the efficacy of these areas are rarely systematically assessed, often as a byproduct of underfunding, particularly in developing countries. Still, adaptive management strategies to maximize conservation success often rely on understanding the temporal and spatial dynamism of population therein. Environmental DNA (eDNA) has been employed as a time and cost-effective method to monitor species’ distribution, with quantitative PCR (qPCR) techniques also assisting in our knowledge about abundance of aquatic taxa. To date however, this novel methodology remains underutilized by conservation managers in assessing populations in protected areas. In this study, we used eDNA concentration of the critically endangered Yangtze finless porpoise (Neophocaena asiaeorientalis asiaeorientalis) to circumscribe population ecology in the Tian e-Zhou National Nature Reserve in Hubei, China. We developed, validated, and optimized a qPCR-based eDNA method and applied this protocol to diagnose the geographical reserve use across seasons. Our results suggest spatio-temporal idiosyncrasies, highlighting previously undescribed site and habitat preferences, and a propensity for post-breeding population dispersal. eDNA thus presents a quick and cost-effective method for assessing population-wide utilization of a protected area and, when accounting for environmental-specific nuances, can prove useful for current and future conservation goals.