Inhalation exposure to the resuspended biological particles from public places can cause adverse effects on human health. In this work, carpet dust samples were first collected from twenty example conference and hotel rooms by a vacuum cleaner. A bipedal robot was then used to simulate children's walking with three different shoes (cotton socks, PVC shoes and EVA shoes) in a hotel room. The particle resuspensions were simultaneously monitored by an aerosol spectrometer. In addition, air samples were also taken using a cyclone liquid impinger operated at 400 L min−1, and further subjected to gene sequencing analysis. Our results showed that dominant bacterial genera in the carpet dusts included those containing respiratory pathogens such as Staphylococcus, Acinetobacter and Pseudomonas. The bacterial structures in carpet dusts were shown different among the samples from hotel and conference rooms (p < 0.05). Robot-walking resuspended a significant amount of particles from the floors, and different shoes have produced different size and concentration level particles (p < 0.05). Furthermore, walking was observed to resuspend more large particles than smaller ones for the studied range (0.3–10 μm). Robot walking induced increases in airborne Acinetobacter and Pseudomonas in breathing zones that were simulated for children. The results demonstrated that particle resuspension by walking was strongly influenced by particle size, biological species (particle properties), and shoe’s sole material. The data from this work provide important information for people especially children aged 1–2 years to protect from resuspension exposure of biological agents when using public floors.