Abstract: This study aims to clarify the evolution of microbial structure during the production of canned edible bird's nests. The thermophilic bacteria were then screened out, according to the safety hazards of canned edible bird's nests. A 16S rRNA high-throughput sequencing technology was combined with traditional culture and isolation to explore the microbial structure in edible bird's nest, including the raw materials, rock sugar, liquid sugar in a boiling pot, towels in the filling workshop, liquid sugar sampled from the filling port, towels in the impurity removing workshop, the steamed bird's nests, and products after filling. The Alpha diversity research showed that the filling significantly increased the content of bacteria, indicating a high-risk process. The heat map combined with Principal Component Analysis (PCA) showed that the same dominated bacterial and similar Structure of the colony were found in both the raw material of rock sugar and the product after filling. The sequencing analysis indicated that the spoilage bacterium (Geobacillus and Bacillus) were also found in both the raw materials of rock sugar and the products after filling. More importantly, the thermophilic bacteria to cause the canned spoilage was introduced by the rock sugar, rather than the production process, according to the first hypothesis that the microorganisms were introduced by the raw materials, and the second hypothesis that the microorganisms were introduced by the process. The reason was that the Bacillus significantly increased in the pipeline transportation of rock sugar in the production of canned edible bird's nests. Five strains of thermophilic bacteria were identified in the raw material of bird's nest, and rock sugar, as well as the rock sugar pipeline transportation, particularly under 55°C microorganism culture and isolation combined with the first generation sequencing. These strains were Bacillus smithii (similarity 99.93%), Bacillus smithii (similarity 99.40%), Bacillus smithii (similarity 99.40%), Bacillus gelatini (similarity 99.27%), Aeribacillus pallidus (similarity 97.43%), and Bacillus licheniformis (similarity 99.47%). This finding can provide a theoretical basis and practical guidelines to prevent thermophilic bacteria contaminations during production, thereby reducing the potential food hazards of canned bird's nests. A strong reference can also be offered to control microorganisms in food production.