The invention discloses a vinegar koji of a Chishui vinegar and a preparation method thereof. The preparation method comprises the following steps: grass powder, Polygonum criopolitanum, kadsura pepper stem, L. fasciculiflora, Senecio scandens, negundo chastetree fruit, and dried tangerine peel are treated separately, the remaining Tiantai radix linderae, dahurian angelica root, Nardostachyos Rootand Rhizome, dark plum, ephedra stem, cassia twig, asarum heterotropoides, pachyrhizua angulatus, balloonflower root, peach kernel, Touerfeng, radix bupleuri, Lysimachia christinae, rust-coloured crotalaria herb with root, akebia stem, mosla dianthera, sinensis diels, walnut leaves, Chinese wingnut leaf, peach leaves, Qingqiugan, Diaoyugan, wheat bran and wheat are subjected to cleaning and drying treatment, the natural plants such as dark plum, peach kernel, grass powder, pachyrhizua angulatus, dahurian angelica root, balloonflower root, dried orange peel, and sinensis diels, as well as wheat bran and wheat are processed into mixed koji, and the above materials are naturally fermented according to a traditional technology, the product contains a variety of microorganisms such as Aspergillus, microzyme, and acetic acid bacteria, the obtained vinegar yield rate is high, a blank comparison test is performed, different formulas and different fermentation processes are used, a traditionalfermentation method is used for the blank, and the quality index of the vinegar koji and the total acid content of the vinegar fermented grains are obtained respectively.