Bioaerosols consisting of biologically originated airborne particles such as microbes, metabolites, toxins, and fragments of microorganisms are present ubiquitously in our living environment. The international interests in bioaerosols have rapidly increased because of their many potential health effects. Thus, accurate and fast detection of total bioaerosols in different environments has become an important task for safeguarding against biological threats and broadening the pool of bioaerosol knowledge. To quickly evaluate the total bioaerosol concentration, we developed a localized surface plasmon resonance biosensor based on succinimidyl-ester-functionalized gold nanoislands (SEF-AuNIs) for quantitative bioaerosol detection. The detection limit of our proposed SEF-AuNI sensors for model bacteria Escherichia coli and Bacillus subtilis can go to 0.5119 and 1.69 cells/mL, respectively. To demonstrate the capability of this bioaerosol sensing technique, we tested aerosol samples collected from Bern (urban station), Basel (suburban station), and Rigi mountain (rural and high altitude station) in Switzerland and further investigated the correlation with endotoxin and PM10. The results substantiated that our SEF-AuNI sensors could be a reliable candidate for total bioaerosol detection and air quality assessment.