The invention provides a simple and effective method for constructing plant long fragment in situ DLO Hi-C sequencing library, which belongs to the technical field of plant three-dimensional genome. Mix with Triton X-100, centrifuge, mix the cell nucleus with the system, digest, connect the digested product with the linker, centrifuge, resuspend the cell nucleus in the system, mix with the connection system after a water bath, and centrifuge, and dissociate the cell nucleus Mix the linked system, uncrosslink, purify, incubate the DNA with the pretreated streptavidin magnetic beads, mix the magnetic beads with the Tn5 transposase system, digest and add adapters, wash away the background, amplify, and enrich DNA fragments to obtain sequencing libraries. When the method provided by the invention is used as the test object, the high-throughput data generated has strong advantages in data signal-to-noise ratio and chromatin interaction identification.