Airborne transmission is much more common than previously thought. Based on our knowledge about SARS-COV-2 (COVID-19) infection, the aerosol transmission routes for all respiratory infections must be reassessed. Thus far, the COVID-19 outbreak has caused catastrophic public health and economic crises, posing a serious threat to the lives and health of people around the world and directing public attention toward the airborne transmission of pathogens. The novel coronavirus transmission in the form of nanoaerosols in a wider range hinders prevention and early warning efforts. As a classical bioaerosol sampler, the Andersen six-stage sampler is widely used in the collection and research of aerosol particles. In this study, the physical and biological collection efficiency of the six-stage sampler was explored by qPCR and colony counting method. Results showed that the physical collection efficiency reached more than 50% when the particle size was larger than 0.75 μm. However, the overall biological collection efficiency was only 0.25%. In addition, fluorescence microscopy and flow cytometry were used to detect the microbial state after sampling, and the results showed that the proportion of the collected live bacteria was less than 15% of the total. This result is of great significance not only for the application of the Andersen six-stage sampler in collecting nanosized bioaerosols, but also provides reference for the selection of subsequent detection technologies for effective collection.