Objective To prepare the standard molecular weight fragment mixtures. Methods Primers were designed to prepare clones which contained different sizes of standard molecular weight fragments. The template used for amplification of insert fragments was the pMD18-T vector. Bacteria culture and plasmid extraction were used to obtain abundant target fragment. Unlabeled DNA fragments were prepared by double digestion of the recombinant plasmids, and the fluorescent adaptor was prepared by annealing with two partial reverse complimentary DNA fragments. The unlabeled fragments and fluorescent adaptor were connected by DNA ligation reaction assisted with T4 DNA ligase. In this way, different sizes of standard molecular weight fragments were prepared. Standard molecular weight fragment mixture was finally prepared by mixing all the fragments together before purification. Results Ten standard molecular weight fragments of different sizes were prepared. The sizes of each fragment are 80bp, 124bp, 194bp, 224bp, 254bp, 304bp, 349bp, 399bp, 424bp and 454bp. The internal standard could accurately determine the size of PCR products amplified with the DNATyper15 kit. Conclusion Using this method, the standard molecular weight fragment mixture which meet the requirements of research and laboratory use was prepared, perfectly providing a new method for preparation of the DNA molecular weight standards. The peaks and the size of the prepared DNA internal lane standard are correct, which can be used to calculate the DNA fragments size in capillary electrophoresis.