Abstract: Provided is a proximity ligation assay-based detection method for a high-order structure (HOS) of an RNA virus, including: mixing an RNA virus with a cross-linking agent, conducting cross-linking under ultraviolet (UV) light, and recovering the RNA virus to obtain a cross-linked RNA virus; extracting RNA of the cross-linked RNA virus; conducting fragmentation on the RNA with RNase III to obtain RNA fragments; ligating the RNA fragments and conducting decrosslinking to obtain decrosslinked RNA fragments; constructing a sequencing library for the decrosslinked RNA fragments; and conducting high-throughput sequencing on the sequencing library, and conducting an RNA HOS analysis on a sequencing result. In the present disclosure, high-efficiency short-distance ligation reaction is used to realize the cross-linking of RNA in virus particles in cell culture or collected supernatant, so as to analyze the HOS of RNA virus genome.