The invention provides a fluorescently label amplification kit for simultaneously amplifying human 27 STR loci. The fluorescently label amplification kit comprises 24 autosomal STR loci, one Y-chromosome STR locus, and two specific amplified primer pairs for gender-identified STR loci. The loci are D3S1358, TH01, D21S11, D18S51, Penta E, D5S818, D13S317, D7S820, D16S539, CSF1PO, D2S1338, D6S1043,D22S1045, D19S433, D1S1656, D12S391, D10S1248, D2S441, vWA, D8S1179, TPOX , FGA, SE33, Penta D, DYS391, Y-INDEL and Amelogenin. The loci contain 20 core loci and four preferred loci as prescribed by the Ministry of Public Security, all sites of mainstream kits currently on the market are covered, the risk of incorrect gender identification due to the deletion of the Y-chromosome can be effectivelyprevented, and the advantages of high discrimination power and high probability of paternity exclusion are achieved.