[Objectives] The species diversity of terrestrial birds serves as a crucial indicator for assessing terrestrial ecosystem health and holds significant implications for formulating biodiversity conservation strategies. However, due to the high vigilance of terrestrial birds and the complexity of their habitats, conventional species diversity survey methods often have low accuracy because individuals are difficult to be observed directly in field surveys. In contrast, the survey methods based on molecular scatology do not require direct sighting and allow accurate species identification through fecal DNA analysis. However, the lack of standardized and quality-controlled procedure limits its application in species diversity research. This study aims to establish a species diversity survey protocol for terrestrial birds based on molecular scatology, covering the entire process from fecal sample collection to data analysis. [Methods] This study focused on the birds of Galliformes in the Xiaozhaizigou National Nature Reserve, Sichuan Province. Field surveys were conducted from 2021 to 2023 via the line transect method, covering 27.7% of the total area of the reserve. The surveyed areas were predominantly evergreen coniferous and broad-leaved forests, with elevations ranging from 1 438 to 3 810 m (Fig. 1). During the survey period, a total of 116 avian fecal samples were collected. Species were identified based on the molecular markers COI and Cyt b. The species identification protocol was optimized by integrating several key steps to enhance the success rate of species identification. These steps included selecting the freshness of samples, extracting avian DNA, and determining the minimum DNA concentration threshold required for identification. The Cochran-Armitage trend test was employed to examine the correlation between fecal sample freshness and species identification success rate. [Results] 1) The success rate of species identification in fresh and semi-fresh avian fecal samples was high (fresh: 100%; semi-fresh: 84.4%), whereas that in the old samples significantly decreased to 45.8%. The Cochran-Armitage trend test revealed a positive correlation between fecal sample freshness and species identification success rate (x² = 21.227, df = 1, P < 0.001). 2) The minimum DNA concentration threshold for species identification using avian feces was 3 mg/L. DNA re-extraction is recommended in the case of the DNA concentration below this value. 3) The intraspecies genetic distances based on the COI gene sequence ranged from 0.001 7 for Tragopan temminckii to 0.012 7 for Lophophorus lhuysii, with a mean of 0.007 7. In contrast, the interspecies genetic distances varied from 0.125 (between Tetraophasis obscurus and Pucrasia macrolopha) to 0.250 (between Chrysolophus pictus and Tragopan temminckii), with an average of 0.177. The interspecies genetic distance was 23 times greater than the intraspecies genetic distance, meeting the “10 × rule”, which states that an interspecies genetic distance greater than 10 times the intraspecies genetic distance can be used to differentiate species (Table 2). Furthermore, the phylogenetic analysis revealed that all species formed monophyletic groups, demonstrating that the established protocol can effectively distinguish bird species (Fig. 3). Ultimately, this study successfully identified 87 samples (75% success rate), encompassing 10 bird species, including 6 species of Galliformes (Table 1), and established a species diversity survey protocol based on avian molecular scatology (Fig. 4). [Conclusion] This study analyzes the relationship between the avian fecal freshness and species identification success rate, and optimizes the species identification protocol through a combination of multiple steps to improve identification success rate. The standardized survey protocol based on molecular scatology provides technical support for bird species diversity monitoring in nature reserves, and can be applied to biodiversity assessment and conservation management.