Objective To establish multiplex system of 16 miniSTR loci, and explore its application value for the degraded materials in forensic medicine. Methods The multiplex system of 16 miniSTR loci was established using a six-dye fluorescence labeling technology and its application value in forensic medicine was assessed. Results A six-dye fluorescence labeling miniSTR amplification kit was developed, which enabled 15 autosomal STR loci, Amelogenin locus and DYS391 to be typed simultaneously. This method showed good specificity and could provide stable and accurate typing results with a sensitivity of 50 pg. This system also provided a good test result for the normal biological sample of actual cases. Conclusion The multiplex system of 16 miniSTR loci has application value for degraded and trace materials with the advantages of high sensitivity and database compatibility, which can be used for forensic casework.