PCR primer was designed according to 16S rRNA conserved sequence of Gluconobacter from the GenBank, and a rapid and sensitive fluorescence quantitative PCR using SYBR Green Ι for Gluconobacter detection in fermented dairy products was established. The established method was then used for the specific detection of a panel of three Gluconobacter strains and 18 non-Gluconobacter strains, with the reliability of the results verified by agarose gel electrophoresis. The results showed that the primers were highly specific. Melting curve analysis of the PCR amplification product showed that primer dimers were present in the amplification product. However, nonspecific amplification could be excluded using the peak time of the melting curve. The standard curve based on Gluconobacter detection using the established method had good correlation (R2 = 0.9968). The minimal detection limit was 75 CFU/mL in the sensitivity test. The established method also successfully detected Gluconobacter in five artificially contaminated samples. These results indicated that the SYBR Green Ι fluorescence quantitative PCR method used herein exhibited high sensitivity and was easy to use. Therefore, this method is suitable for the qualitative detection of Gluconobacter strains in fermented dairy products.