A duplex-seq-based ultra-low frequency mutation site detection and analysis method disclosed in the present invention comprises the following steps: 1) evaluating the quality of original sequencing data, reducing data noise, and providing effective data for subsequent analysis; 2) random barcode Extract the title line of each sequence of the sequence file, which is convenient for subsequent quick retrieval of barcode and creation of consistent sequences; 3) Create consistent sequences based on family barcode and duplex barcode, and exclude mutations introduced during the library construction process or PCR process ; 4) Construct a double-stranded consensus sequence according to duplex-tag, and further exclude asymmetric mutation sites in the sequence; 5) Perform local quality correction on the compared data, and perform low-frequency mutation site detection; Three-level annotation of gene structure, function, and clinical phenotype; 6) Statistics of SSCS, DCS sequence numbers, comparison results, and variation site information, and output visual charts.