The invention discloses a fluorescent labeled X-STR locus multiplex PCR method and an application thereof; the system performs multiplex amplification analysis of 12 loci: GATA172D05, DXS6789, DXS10074, DXS10078, GATA165B12, DXS6797, DXS6803, DXS6804, GATA31E08, DXS7130, DXS9895, and DXS6810, wherein primers of the 12 loci are labeled respectively by four fluorescences of FAM, HEX, TAMRA, ROX. The method of the invention can be used to prepare a set of kit as an X-STR locus multiplex amplification kit with Chinese characteristics, and the kit is applicable to gene localization of paternity identification, individual identification, sex identification, and X-linked genetic diseases, and is especially applicable to paternity identification for sister recognition, half-blood half-sister recognition, generation-skipping recognition, etc.