The invention provides a preparation method of a DNA (Deoxyribose Nucleic Acid) gelatin nano-microgel anti-counterfeit coating. The preparation method comprises the following steps of: mixing DNA with a gelatin solution, then precipitating through a precipitant, cross-linking and solidifying to obtain nano-microgel so that the DNA is wrapped with a water-containing gelatin nano-microgel network; and then adding such gelatin nano-microgel to the coating in a certain proportion, thus obtaining the anti-counterfeit coating which is long in storage period, stable in structure, easily detected, natural and nontoxic. According to the preparation method, a primer is designed according to special DNA composition and in light of the unknown performance and amplification performance of the DNA, the polymerase chain reaction (hereinafter referred to as PCR) is carried out to realize amplification by million times, then the contained DNA can be detected by electrophoretic analysis, and therefore, the anti-counterfeit purpose is realized.