Timely detection of harmful bioaerosols is critical for personnel protection and quick operational decisions. Conventional detection and identification methods are often slow, costly and impractical for onsite analysis, limiting early intervention and disease containment. This work integrates electrochemical impedance spectroscopy (EIS) with digital microfluidic (DMF) technology to enable rapid, multiplexed detection of pathogens using Toll-like receptor (TLR) proteins as biorecognition elements. This low-burden, portable broad-spectrum sensor could distinguish diverse airborne pathogens in near-real time without the need for a priori knowledge.   The integrated EIS-DMF detection platform consists of multiplexed electrodes functionalized with different TLRs to recognize specific, immutable pathogen-associated molecular patterns. Pathogen simulants were aerosolized in controlled and field conditions, collected using commercial and custom biosamplers, and analyzed on the DMF device. Impedance changes before and after sample exposure indicate the presence and types of pathogens, and the results were validated against quantitative PCR and lateral flow assay measurements.   The multiplexed and integrated EIS biosensor successfully detected and differentiated aerosolized pathogen simulants within 30 minutes. For example, TLR2/6 sensors responded distinctly to Bacillus atrophaeus spores, enabling qualitative comparison of biosampler performance. Additionally, bioaerosol sample analyses confirmed measurable signal changes above background levels, validating the sensor’s capability for near-real-time, in-field detection and classification of airborne pathogens.   These findings represent a significant advancement in portable, multiplexed biodetection technologies. This approach bridges the gap between laboratory-based diagnostics and field-deployable environmental monitoring tools, supporting rapid, broad-spectrum sensing platforms that strengthen public health preparedness, defence and security through early detection of emerging microbial threats.