Submerged macrophytes and phytoplankton assemblage play significant roles in the functioning of aquatic ecosystems. An experiment was carried out in Beijing in order to further evaluate the environmental factors that affect the growth of submerged macrophytes and phytoplankton assemblage. Submerged macrophytes (i.e., Vallisneria natans, Hydrilla verticillata, and Ceratophyllum demersum) constructed the growth system with some controllable influencing factors (i.e., the flow rate and water depth gradient). The flow rates were set separately as 4L/h (1#), 6L/h (2#), and 12L/h (3#), while the water depth gradient was of 0.5-1.7m in eutrophic water. Generally, all macrophytes could grow normally in the experiment, and the system could maintain and improve the effluent quality. The average removal rates of NH3-N, COD, NO3-N, TN, and TP were about 90%, 33%, 65%, 45%, and 40%, respectively. Seen from the results of the water depth gradient experiments, it is indicated that Vallisneria natans grows better in shallow water (0.5m) and moderate shallow water (0.7m) area, with an average relative growth rate (ARGA) of 57%. Hydrilla verticillata and Ceratophyllum demersum grow better in moderate deep water (1.2m) and deep water (1.7m) area (ARGA of 66% and 64%, respectively). Results of the flow rate experiments showed that the moderate flow rate (6L/h) was the best for those three macrophytes' growth. As the fitting results of the rapid light curves (RLCs) showed that the utilization of light and the tolerance to strong light were different for these macrophytes, if they are ranked in the order of the utilization and the tolerance from strong to weak, they are Hydrilla verticillata, Ceratophyllum demersum, and Vallisneria natans. Microbial analyses indicated that the overall system diversity of the experimental groups have been improved after cultivation of macrophytes. However, the accumulated Cyanobacteria caused by the low flow rate (1#) would lead to the suppression of microbial organics decomposition and nutrient metabolism in the macrophytes. To sum up, the results of this study provided theoretical guidance and technical support for the restoration of submerged macrophytes in eutrophic water.