The invention provides a multiplex amplification system based on rapid mutation Y-STR gene loci, a method and application. The multiplex amplification system comprises 13 gene loci, namely, DYF387S1, DYF399S1, DYF403S1a/b, DYF404S1, DYS449, DYS518, DYS526b, DYS547, DYS570, DYS576, DYS612, DYS626 and DYS627, wherein sequences of amplification primer pairs are shown as SEQ ID NO.1-26, and the gene loci are marked through four fluorescein of 6-FAM, VIC, NED and PET respectively. According to the multiplex amplification system based on the rapid mutation Y-STR gene loci, the method and the application, for the 13 rapid mutation Y-STR gene loci, the primers are designed on the basis of group frequency survey, and the gene loci are integrated into the multiplex reaction system according to specific combination and primer concentration, so that amplification and detection of the gene loci are achieved simultaneously, and the amplification effect is better; when the multiplex amplification system is applied to the detection process, convenience and rapidness are better achieved, the typing result is clear, the correctness, sensitivity and stability of gene locus detection can be guaranteed, and higher individual recognition rate is achieved.