The 100 bp gradient DNA molecular weight marker and its preparation belongs to the field of molecular biology reagent preparing technology. The molecular weight marker is one kind of mixture comprising 11 DNA stripes with the lengths of 100 bp, 200 bp, 300 bp, 400 bp, 500 bp, 600 bp, 700 bp, 800 bp, 900 bp,1000 bp, and 1200 bp separately and the concentration of 4 ng / microliter. The preparation process includes simultaneous synthesis of the 11 DNA stripes through PCR with 11 pairs of specific primers and plasmid PBR322 as template DNA in the presence of Taq DNA polymerase, Taq DNA polymerase buffering liquid and 4 kinds of ribotides (dTTP, dATP, dCTP and dGTP), in the same amplification condition in a PCR instrument; and the subsequent purification and mixing. The present invention features the fast, precise and mass preparation of DNA molecular weight markers of different lengths in the set target.