Protected areas are key to meeting biodiversity conservation goals, but direct measures of effectiveness have proven difficult to obtain. We address this challenge by using environmental DNA from leech-ingested bloodmeals to estimate spatially-resolved vertebrate occupancies across the 677 km 2 Ailaoshan reserve in Yunnan, China. From 30,468 leeches collected by 163 park rangers across 172 patrol areas, we identify 86 vertebrate species, including amphibians, mammals, birds and squamates. Multi-species occupancy modelling shows that species richness increases with elevation and distance to reserve edge. Most large mammals (e.g. sambar, black bear, serow, tufted deer) follow this pattern; the exceptions are the three domestic mammal species (cows, sheep, goats) and muntjak deer, which are more common at lower elevations. Vertebrate occupancies are a direct measure of conservation outcomes that can help guide protected-area management and improve the contributions that protected areas make towards global biodiversity goals. Here, we show the feasibility of using invertebrate-derived DNA to estimate spatially-resolved vertebrate occupancies across entire protected areas.