The invention discloses an ultra-low frequency mutant nucleic acid fragment detection method, a mutant nucleic acid fragment library construction method, a primer design method and a reagent. Firstly,the fragmented nucleic acid of the sample to be tested is subjected to terminal repair, end-filling and adding A; connecting molecular tag joints; using the combination of molecular tag primers and single molecule specific primers to carry out amplicon targeted enrichment on the connection jointer product; finally, performing PCR amplification and enrichment. The single molecule specific primer designed by the invention obviously improves the number of available effective templates, and the carried adjustment sequence can obviously reduce the probability of forming primer dimers among specific primers, and the utilization rate of templates can be also obviously improved. The ultra-low frequency mutation nucleic acid fragment detection method of the invention uses one-sided single primer amplicon enrichment method and molecular label technology, improves the utilization rate of effective templates, reduces noise sources, reduces false positives, improves detection sensitivity and specificity, and is especially suitable for detection of trace ultra-low frequency mutation samples. At the same time, the invention can also carry out high-efficiency library construction.