The safety of water supply network has always been a concern. The biofilm attached on the pipes will fall into pipe-water under the disturbance, which becomes a potential threat towards human health. Particulate matter can provide shelter for bacteria, and most bacteria in water will adhere to the surface of it, which is not easy to be killed by disinfectants. Therefore, the effect caused by particulate matters and extracellular polymer (EPS) on the stability of biofilm was investigated. This study revealed that the biofilms without particulate matters showed the highest biomass, that’s an order of magnitude more than have particulate matter bacteria, while those with abundant particulate matters and bacteria were more stable. The effect of shear stress towards the biofilm attached on the pipes was investigated through rotational speed experiments, and the results implied that the collision between bacterial polymers increased with the increasing of rotational speed, simultaneously with the formation of stale biofilm adhered inside the pipes. Besides, instead of protein and fatty chains, the hydroxyl bonds played vital roles during the combination of particulate matters and EPS.