The presented investigation focused on exploring the characteristics of the biofilm formed on a novel semi-suspended bio-carrier and revealing their variation during the whole growing cycle. This used semi-suspended bio-carrier was designed to be a spindle shape, and then fabricated by using a 3D printing technique. Results indicated the bio-carrier provided a suitable environment for the attachment of diverse microorganisms. During the experimental period lasted for 45 days, the biofilm quickly attached on the surface of the bio-carrier and grew to maturity, but its characteristics, including the chemical compositions, adhesion force, surface roughness, structure of microbial communities, varied continuously along with the operational time, which greatly influenced the performance of the bioreactor. The shape and structure of bio-carrier, and the shearing force caused by the aeration are important factors that influence the microbial community and its structure, and also heavily affect the formation and growth of biofilm.