Bioaerosols constitute a major route of disease transmission, necessitating effective detection methods for public health protection. In this study, a microfluidic colorimetric biosensor integrated with the water-soluble nonwoven fabrics (WS-NWFs) was innovatively developed for the collection and detection of bacterial aerosols. The WS-NWFs made of poly(vinyl alcohol) with good biocompatibility and water solubility were used to collect the bacterial aerosols, which eliminated the need for mechanical or chemical elution, enabling direct release of collected bacteria, thereby maximizing the recovery rate. The immune magnetic nanobeads (MNBs) and Pd/Pt nanozymes were employed to specifically separate and label the collected bacteria, achieving enrichment of the collected bacteria and amplification of the biological signal. Meanwhile, the microfluidic colorimetric biosensor with a multivortex micromixer integrated bacterial separation, labeling, washing, catalysis, and detection, realizing the quantitative determination of bacterial aerosols. The proposed biosensor provides a rapid, specific, sensitive, and in-field solution for real-time monitoring of pathogenic bioaerosols in public health settings.