<p>Microbial oil and gas detection technology is a nonseismic technique for predicting the occurrence and distribution of oil and gas accumulation at greater depths through the detection of indicator microbes in surface soil or offshore sediment samples. As a surface technology, it is affected by landforms, soil physical and chemical properties, the heterogeneity of microbial distribution and other factors, thus affecting oil and gas prediction. This study challenges the traditional single-point and single-sample collection method for microbial sample collection. It attempts to combine collection methods to improve the representativeness of samples. To quickly obtain onsite microbial detection results and monitor the quality of sample collection, rapid microbial screening technology was developed. In addition, methods for identifying and correcting surface environmental factors were explored. The results suggest that the mixed collection method and three samples averaged from a single point have similar effects and are effective at improving the representativeness of soil samples. A microbial quick screening culture system with an optimal butanol concentration of 0.4% and a reaction time of 14–22 h was developed. In practice, conclusions similar to those from conventional laboratory plate culture methods were obtained. By adjusting the sampling depth, changing the dilution for sample detection, and processing data between zones, it is possible to more accurately characterize subsurface oil and gas seepage, thereby improving the reliability and validity of the microbial data.</p>