The invention relates to a Hi-C high-throughput sequencing library building method suitable for watermifoil. The method includes the steps of firstly, performing formaldehyde crosslinking on a watermifoil sample to obtain a crosslinking material; secondly, performing cell lysis to release chromatin; thirdly, collecting the chromatin, and sequentially subjecting the chromatin to SDS deactivation endogenous enzyme and MboI endonuclease digestion breaking to obtain a material after digestion; fourthly, performing tail end filling to obtain tail-end-filled DNA, and performing intramolecular connection on the tail-end-filled DNA; fifthly, decrosslinking the intramolecular connection material and purifying DNA, and performing DNA breaking to form fragments beneficial to sequencing; seventhly, building a Hi-C library for high-throughput sequencing; eighthly, specifically capturing biotin marked DNA fragments, and performing amplification; ninthly, performing library quality inspection. The method has the advantages that the appropriate cell breaking method, formaldehyde crosslinking condition, endogenous enzyme deactivation condition and appropriate tool enzymes are designed according tothe special structure of watermifoil cells, the problem that the cell wall structure of the watermifoil affects a Hi-C experiment, Hi-C high-throughput sequencing library building of the watermifoil is achieved, and the application range of the Hi-C technology is expanded.