[Objective] We developed a rapid SYBR Green I fluorescent quantitative real-time PCR technique for detection of methane-oxidizing bacteria for microbial prospection of oil and gas. [Methods] The recombinant plasmid containing pmoA gene was used as standards to optimize the experimental conditions and generate a standard curve. The sensitivity, reproducibility and specificity of the technique were evaluated, and practical soil samples were detected using the technique. [Results] The correlation coefficient (R2) of the standard curve was 0.999 9 and the amplification efficiency was 99.976%. The detection range was from 3.897×101 to 3.897×109 copies/μL and the sensitivity was about 40 copies/μL. All variable coefficients of CT values in the reproducibility test were better than 3%. In addition, no amplification was observed in the templates of 12 non-methane-oxidizing bacteria. The technique showed good sensitivity, specificity and reproducibility. Soil samples collected in the gas field, the oil field and the non-oil and gas field were detected using the technique, and the gas field had high anomalies of methane-oxidizing bacteria. [Conclusion] The fluorescence quantitative real-time PCR technique developed here could rapidly and exactly detect methane-oxidizing bacteria for oil and gas exploration. At the same time, this technique could contribute to detection of other indicator bacteria in oil and gas field soil.