Clarifying the distribution pattern and overlapping relationship of sympatric relative species in the spatio-temporal niche is of great significance to the basic theory of community ecology and integrated management of multi-species habitats in the same landscape. In this study, based on a 9-year dataset (2012–2021) from 493 camera-trap sites in the Gongga Mountain National Nature Reserve, we analyzed the habitat distributions and activity patterns of tufted deer (Elaphodus cephalophus) and sambar (Rusa unicolor). (1) Combined with 235 and 153 valid presence sites of tufted deer and sambar, the MaxEnt model was used to analyze the distribution of the two species based on 11 ecological factors. The distribution areas of the two species were 1038.40 km2 and 692.67 km2, respectively, with an overlapping area of 656.67 km2. Additionally, the overlap indexes Schoener’s D (D) and Hellinger’s-based I (I) were 0.703 and 0.930, respectively. (2) Based on 10,437 and 5203 independent captures of tufted deer and sambar, their daily activity rhythms were calculated by using the kernel density estimation. The results showed that the daily activity peak in the two species appeared at dawn and dusk; however, the activity peak in tufted deer at dawn and dusk was later and earlier than sambar, respectively. Our findings revealed the spatio-temporal niche relationship between tufted deer and sambar, contributing to a further understanding of the coexistence mechanism and providing scientific information for effective wild animal conservation in the reserve and other areas in the southeastern edge of the Qinghai–Tibetan Plateau. © 2022 by the authors.