Abstract • Sampling parameters with high efficiency was determined. • Operational process to detect airborne ARGs was optimized. • Providing research basis to control airborne ARGs of a laboratory atmosphere Antibiotic resistance genes (ARGs) have been detected in various atmospheric environments. Airborne ARGs transmission presents the public health threat. However, it is very difficult to quantify airborne ARGs because of the limited availability of collectable airborne particulate matter and the low biological content of samples. In this study, an optimized protocol for collecting and detecting airborne ARGs was presented. Experimental results showed that recovery efficiency tended to increase initially and then declined over time, and a range of 550–780 copies/mm2 of capture loading was recommended to ensure that the recovery efficiency is greater than 75%. As the cell walls were mechanically disrupted and nucleic acids were released, the buffer wash protects ARGs dissolution. Three ratios of buffer volume to membrane area in buffer wash were compared. The highest concentrations of airborne ARGs were detected with 1.4 µL/mm2 buffer wash. Furthermore, the majority of the cells were disrupted by an ultrasonication pretreatment (5 min), allowing the efficiency ARGs detection of airborne samples. While, extending the ultrasonication can disrupt cell structures and gene sequence was broken down into fragments. Therefore, this study could provide a theoretical basis for the efficient filter collection of airborne ARGs in different environments. An optimized sampling method was proposed that the buffer wash was 1.4 µL/mm2 and the ultrasonication duration was 5 min. The indoor airborne ARGs were examined in accordance with the improved protocol in two laboratories. The result demonstrated that airborne ARGs in an indoor laboratory atmosphere could pose the considerable health risk to inhabitants and we should pay attention to some complicated indoor air environment.