The invention belongs to the technical field of bioinstrumentation, in particular to a fluorescent-marked 16 X-STR loci multiplex amplification system and application thereof. The system performs multiplex amplification analysis on 16 X-STR loci, namely GATA165B12, DXS101, GATA172D05, HPRTB, DXS981, DXS8378, DXS6795, GATA31E08, DXS6809, DXS6803, DXS9902, DXS6807, DXS7423, DXS7133, DXS6810 and DXS7132; the 16 loci are independent relatively and not linked mutually; and corresponding primers are respectively marked by four fluorescent markers, namely FAM, HEX, TAMRA and ROX. Based on the multiplex amplification system, an X-STR loci multiplex amplification kit suitable for Chinese people can be prepared for parentage analysis, individual recognition, sex tests, and gene location of X-linked inheritance disease, particularly for paternity identification of sisters, blooded half-sisters and skipped generations.