Objective To develop an eight-dye fluorescent multiplex amplification system based on mini-STR and eight-dye fluorescent labeling technique, and to evaluate its applicability in the analysis of degraded samples in forensic medicine. Methods An eight-dye fluorescent labeling technique was used to develop a multiplex amplification system targeting 26 loci, including D22S1045, D13S317, CSF1PO, D2S441, D10S1248, TPOX, D16S539, D5S818, Penta D, TH01, D2S1338, Penta E, rs2032678,Amelogenin, D19S433, D21S11, D6S1043, D8S1179, vWA, D7S820, D1S1656, DYS391, D18S51, D12S391, D3S1358, and FGA. The established amplification system was evaluated and tested for its specificity, sensitivity, accuracy, and the ability to analyze degraded samples. Results An eight-dye fluorescent multiplex amplification system containing 26 loci was successfully developed. All amplicons were less than 310 bp in length. The system demonstrated high specificity, sensitivity, and accurate typing, and performed well in the analysis of degraded samples. Conclusion The mini-STR with the eight-dye fluorescent multiplex amplification system has good potential for the analysis of degraded samples and can be applied to forensic casework.