Abstract: The nature reserve is the material carrier and important support of the development on ecotourism activities and the construction projects will inevitably have an impact on nature reserves. Therefore, it is necessary to evaluate the impact on biodiversity in nature reserves of ecotourism construction projects. Taking the existing ecotourism construction project in Wolong Valley Scenic as an example, the natural landscape types, vegetation, characteristics of plant communities, animal and plant species and distribution characteristics were investigated and evaluated through field investigation and literature review. According to the Technical Regulation for Biodiversity Impact Assessment of Construction Project in Nature Reserve (LY/T 2242—2014), the biodiversity in Wuyuan Forest Bird National Nature Reserve in Jiangxi Province was evaluated from six aspects, including landscape/ecosystem, biological community, population/species, main protection objects, biosafety and social factors. The results showed that the diversity impact index of construction projects on the protected area was 53.98, which was a medium-low degree of impact. At the same time, combined with the existing ecotourism construction projects and actual situations of the reserve, the measures to mitigate the impact and protection suggestions were put forward to further reduce possible negative impacts.