A helicase-dependent isothermal DNA amplification (HDA) assay was developed for the rapid and accurate detection of Gluconacetobacter in yogurt.A pair of specific oligonucleotide primers according to the 16S ribosomal RNA gene (GenBank accession number:HQ677466.1) of Gluconacetobacter was designed,and the optimal concentration of UvrD helicase and T4 gp32 in the reaction system were determined.The HAD method enabled direct detection of Gluconacetobacter in yogurt.The specificity of the method was tested by amplification and electrophoresis of various strains in yoghurt using the HAD system.The amplification product by HDA was the same as the designed gene fragment (101 bp).The sensitivity of HDA was 102 CFU/g.The optimal reaction system was established by the use of 0.1 μg of UvrD helicase and 5.0 μg of T4 gp32.In conclusion,the method was sensitive and time saving.