Expressmarker mtDNA-SNP60 kit, a 3-dye fluorescence labeling forensic mitochondrial-SNP kit, was developed and modified based on previous work. In this study, it was validated through sensitivity, precision, accuracy, reproducibility, species specificity, mixture samples, PCR-based conditions, and inhibitor studies, according to the Scientific Working Group on DNA Analysis Methods guidelines. In the sensitivity study, full profile was obtained from template DNA with the quantity >= 4 pg. Consistent profiles were obtained at three different laboratories, which demonstrated the reproducibility of the kit. No peak was observed in the profiles of common animal species and microorganisms demonstrating high species specificity of the kit. In the mixture study, some loci (minor contributors) were missing at 19: 1 and 9: 1 ratios, whereas all loci could be profiled at other ratios (4: 1, 2: 1, and 1: 1). PCR-based studies demonstrated that there recommended PCR conditions were optimized for the kit, and there was acceptable tolerance of error. The inhibitor study indicated that the kit has varying degrees of resistance to the presence of common inhibitors. Twelve random individuals were profiled by next-generation sequencing to confirm the allele-specific PCR assay, and a consistent result was obtained. Furthermore, 520 individuals from 260 families were profiled and the results showed that all loci of the confirmed mother and child were matched. In addition, forensic population genetics parameters, including random match probability, discrimination power, and haplotype diversity, were calculated (0.0474, 0.9526, and 0.9563, respectively). Our study demonstrates that the Expressmarker mtDNA-SNP60 kit can be a useful supplementary tool for forensic application.