A rapid method using helicase-dependent isothermal DNA amplification (HDA) for the detection of Gluconobacter in fermented milk was established. The ITS gene sequence (gene number KF896260.1) of Gluconobacter was selected as the target gene for design of specific primers, and the concentrations of UvrD helicase and T4 gp32 in the reaction system were optimized to establish an optimal reaction system. The HDA method was used to directly detect Gluconobacter in fermented milk. The established HDA system was used to amplify and electrophoretically detect various strains in fermented milk to verify the specificity of the method and determine its detection limit. The results showed that the detection of Gluconobacter in fermented milk by the HDA method was consistent with the designed sequence length (309 bp) and the detection limit was 4.3 × 101 CFU/g. The appropriate amounts of UvrD helicase and T4 gp32 added in the reaction system were 0.1 and 5.0 μg, respectively. The method is highly sensitive and time saving.