Bio-aerosol concentrations in poultry houses must be controlled to provide adequate air quality for both birds and workers. High concentrations of airborne bio-aerosols would affect the environmental sustainability of the production and create environmental hazards to the surroundings via the ventilation systems. Previous studies demonstrate that several factors including the age of the birds, the housing configuration, the humidity and temperature would strongly affect the indoor concentration of bio-aerosols. However, limited studies are performed in the literature to investigate the bio-aerosol dispersion pattern inside poultry buildings. In order to fill a gap of the understanding of the bio-aerosol dispersion behavior, experimental measurements of the indoor bio-aerosol distribution are performed in a tunnel-ventilated poultry house in this paper. Meanwhile a three-dimensional computational fluid dynamics (CFD) model is built and validated to further investigate the effect of flow pattern, turbulence and vortex on the dispersion and deposition of the bio-aerosols. Furthermore, bio-aerosols with various diameters are also examined in the CFD model. It is found that higher concentrations of bio-aerosols are detected at the rear part of the house and strong turbulent flow resulting from the ventilation inlets enhances the diffusion and dispersion of bio-aerosols. Local vortex or disturbed flow is responsible for higher local concentration due to the re-suspension of settled bio-aerosols, which suggests that careful attentions should be paid to these locations during cleaning and disinfection. Results from present study contribute to the optimization of design and operation of the poultry houses from the standing point of reducing airborne bio-aerosol concentrations.