In this study, a novel EBC collection method was developed by using hydrophobic surface, a layer of ice, and a droplet scavenging procedure. The physical collection efficiency (amount of EBC collected per unit of time) of the device was evaluated. In addition, biological analysis and characterization of EBC samples collected from human subjects were conducted using culturing, DNA stain, SEM, qPCR and species identification tool VITEK 2. This work contributes to the effort in applying EBC together with molecular tools as a non-invasive method in rapid disease diagnosis.