Reusing reclaimed water for the ecological water has become one of the effective ways to alleviate the water crisis in the rivers and lakes, while its impact on the river and lake ecosystems has become the focus of public attention. The microorganism is one of the most direct and effective monitoring objects, which is generally present in the form of biofilm. Therefore, three kinds of water with different water quality were chosen, which included reclaimed water treated with anaerobic–anoxic–oxic (A2/O) and cyclic activated sludge system process, and surface water, respectively. Glass plates with different roughness (0.1, 1.0 and 10 μm) were used as attaching matrix. Biofilms were cultured with the help of outdoor simulation environment; and the dynamic variation of the biofilm dry weight as well as inorganic and organic components were analyzed. The results showed that the content for dry weight, iron oxide, extracellular polymeric substances (EPS) and chlorophyll a of biofilms attached on multi-media surface in three kinds of water body increased sequentially during the rapid growth period, while the activity of phosphatase fluctuated. Biofilm dry weight and chlorophyll a variation can be described double-constant rate equation (R2 is over 0.90 and 0.91, respectively), while the iron oxide and EPS content could make use of the parabolic diffusion equation (R2 is over 0.88 and 0.87, respectively). For the surface of the medium with different roughness, dry weight of the different biofilms with roughness 1.0 μm has the largest content, up to 1.39 mg cm−2. With regard to iron oxide, the content differences are little with different roughness and the maximum content is up to 0.36 μg cm−2, while no significant difference existed between the different water bodies. The contents of EPS, chlorophyll a and phosphatase activity in reclaimed water are higher than that in the surface water, and both of them have the highest content with the roughness 1.0 μm medium surface. Testing phospholipid fatty acids showed that the dominant bacteria are i15: 0, bacteria under two processes of reclaimed water in biofilms were, respectively, higher by 3.7 and 3.5 % than the surface water. Biofilm EPS and chlorophyll a concentration was positively correlated with pH, total phosphorus, COD and Mg2+ concentration in the reclaimed water, and was negatively correlated with NH4+, while the phosphatase activity showed no correlation with water quality indicators. This research aimed to provide a theoretical basis and technical support for health assessment of reclaimed water as the ecological water ecosystem for urban rivers and lakes.