The response of Vallisneria natans to the increase contents of nitrogen and phosphorus were studied under laboratory conditions by measuring chlorophyll fluorescence parameters of V. natans using a pulse-amplitude modulated fluorometer (Diving-PAM), combined with measuring some other physiological indexes of V. natans. The results showed that V. natans responded quickly to water nitrogen and phosphorus concentration changes, the maximum quantum yield decreased significantly after 2 h and 6 h, the maximum quanta yield returned to normal levels 12 h after the treatment. There was no significant difference between treatment groups for the maximum quantum yield. Inhibitory effect of light intensity on photosynthesis of V. natans was more obvious under higher (treatment D) or lower (treatment A) concentrations of nitrogen and phosphorus. The average quantum yields of four measurements in the treatment B or C was significantly higher than that of A or D (p < 0.05). Within a certain range, chlorophyll content increased significantly with the nitrogen and phosphorus concentrations. When nitrogen and phosphorus concentrations further increased and reached the level of D treatment, chlorophyll content of V. natans decreased. Malondialdehyde (MDA) content of V. natans changed in the different treatment groups, MDA content of treatment B was the lowest while treatment D was the highest. It shows that V. natans is more suitable for growing in eutrophic waterbodies, but excessive nitrogen and phosphorus could inhibit the physiological activities of V. natans.