Environmental DNA (eDNA) techniques show promise in facilitating the study and conservation of threatened species. The advantages of high sensitivity and high efficiency have been demonstrated by studies across various taxa. Asian turtles, one of the most threatened vertebrate groups, are facing extinction due to overexploitation for use as traditional Chinese medicine, food, and pets. Most species are extirpated across their ranges, but Hong Kong is unique because wild turtle populations persist. Due to the rarity and secretiveness of turtles, data on the location and size of populations are incomplete, even for a small place like Hong Kong. The result is that research and conservation are hindered. In this study, quantitative PCR eDNA assays were developed for three turtle species: two native [big-headed turtle (Platysternon megacephalum) and Chinese softshell turtle (Pelodiscus sinensis)] and one invasive [red-eared slider (Trachemys scripta elegans)]. For each assay, we tested species specificity, optimized reactions, and benchmarked analytical sensitivity for eDNA surveys. The assays will help in the conservation of Hong Kong turtles by identify priority sites for protection and research, provide another tool for population monitoring, and clarify the impact of the invasive T. s. elegans on native species.