Submerged macrophytes are key biological components in maintaining the clear water state of shallow lakes and structure and function of lake ecosystems. In recent years, the ongoing climate change and increasing human activities have led to prominent changes in the hydrological regime of lakes, having major influences on growth, reproduction, and succession of submerged macrophyte species. In this study, we set up two water level fluctuation amplitudes and two fluctuation frequencies, with constant water level as a control, to explore the responses of biomass and above- and belowground morphological traits of the submerged macrophyte Vallisneria spinulosa to water level fluctuations. Our results showed that compared with the constant water level, fluctuating water level treatments (small amplitude and low frequency, small amplitude and high frequency, large amplitude and low frequency, and large amplitude and high frequency) had a significant effect on total biomass and the resource-harvesting structures of V. spinulosa. Compared with the control, water level fluctuations significantly decreased the number of ramets, leaves and roots, length of roots, number and total length of stolons, as well as leaf, root, above- and belowground biomass. However, water level fluctuations obviously enhanced plant height and specific leaf area. Both frequency (low and high) and amplitude (small and large) of water level fluctuations had no significant effect on morphology of aboveground photosynthetic organs, but the biomass accumulation of leaves and the aboveground part were significantly affected by the amplitude of water level fluctuations. Furthermore, both the number and length of roots were affected by amplitudes but not by frequencies of water level fluctuations. Therefore, it is necessary to avoid large range of water level fluctuations during the V. spinulosa community reestablishment in shallow lakes, in order to enhance the recovery ability of submerged macrophytes.