The invention relates to a Hi-C high-throughput sequencing library building method suitable for microbial metagenomics and application. The sequencing library building method comprises the following steps that 1), a metagenome sample is taken to be subjected to microbial and impurity separation, and formaldehyde cross-linking is carried out; 2), endonuclease digestion disruption is carried out oncell chromatin to obtain a material after endonuclease digestion; 3), end filling is carried out on the material after endonuclease digestion; 4), DNA cell nucleus intramolecular connection is carriedout; 5), unconnected end biotin is removed to obtain purified DNA, and the purified DNA is subjected to fragmenting, end repairing and A-tailing, and ligation adaptor; 6), DNA target fragments are sorted; and 7), the target fragments are captured by biotin, and library amplification and sequencing are carried out. According to the Hi-C high-throughput sequencing library building method suitable for microbial metagenomics and the application, microorganisms in a complex environment are enriched through separation of the microorganisms and environmental impurities, metagenome data analysis is not limited to a single species any more, and various microorganisms in the complex environment can be subjected to clustering analysis, so that Hi-C high-throughput sequencing library building of microbial metagenomics is achieved, and the application range of the Hi-C technology is expanded.