The invention discloses a complex amplification kit for supplementing STR site for difficult detection materials and application thereof. The kit comprises 17 pairs of specific amplification primers of STR loci, wherein the STR loci are Amelogenin, D3S1358, D6S477, D1S1656, D19S253, D2S441, D6S1043, D15S659, D3S3045, Penta E, D8S1132, Penta D, D10S1435, D22S1045, D10S1248, D12S391 and DYS391. Thekit has the advantages of rapid and efficient detection of the difficult detection materials such as trace, degradation and inhibitor-containing materials in a field case; and meanwhile, the kit has high efficiency for amplification of the difficult case detection materials, and the optimized locus information can supplement the existing case database, shorten the case investigation time and lockthe suspect.