Eating bird′s nest (EBN)can regulate intestinal flora and improve human immunity. Excavating the key nutrients and effective functional groups in fresh stewed bird′s nest (FSBN)can lay a research foundation for exploring the mechanism of bird′s nest regulating intestinal flora. The functional groups and material composition of the two brands of FSBN products were analyzed by Fourier Transform Infrared Spectroscopy (FTIR) and high-resolution mass spectrometry (HRMS), respectively. In order to explore the effective functional groups and key regulatory substances in the regulation of intestinal flora after eating FSBN, the relationship between FTIR and HRMS results and the abundance of intestinal flora in C57BL/6N mice was established by correlation analysis. The results showed that after taking FSBN, the intestinal bacteria that differ greatly in the species level are as follows: beneficial bacteria Lactobacillus and Lachnospiraceae_NK4A136_group showed an upward trend, beneficial bacteria Faecalibaculumand Akkermansia showed a downward trend, and harmful bacteria Desulfovibrio, Enterorhabdus and Candidatus_Saccharimonas showed a downward trend. The infrared difference spectra of the two products were mainly concentrated in the 1 700～1 200 cm-1 band, which were mainly the distribution areas of the amide bond, aromatic CC and carboxylic acid CO group. These functional groups were positively correlated with beneficial bacteria Lactobacillus and Lachnospiraceae_NK4A136_group and negatively correlated with harmful bacteria Desulfovibrio and Enterorhabdus. After analysing small molecular substances in FSBN by metabolomics, it was found that the main components were amino acids, lipids, esters and sialic acids, which showed different correlations with intestinal bacteria. Generally speaking, lipids, especially some phosphate esters (Phosphate, Phosphatidylcholine, Phosphatidylethanolamine and Phosphatidylglycerol) and fatty acyl showed extremely significant negative correlation with harmful bacteria and a positive correlation with beneficial bacteria. The characteristic groups in these substances can also correspond to the FTIR results. The above results show that both Fourier transform infrared spectroscopy and metabolomics can provide a theoretical basis for studying nutrients on the regulation of intestinal flora.