In this study, the root exudates of nitrogen-removal efficient Spirodela polyrrhiza(HZ1) and nitrogen-removal inefficient Lemna minor (WX3) from Tai Lake region were collected continuously and separated, and the effects of dose and composition of root exudates on the nitrogen removal efficiency of the denitrifying bacterium Pseudomonas fluorescens were also investigated. The results showed that the nitrogen removal efficiency of P. fluorescens was significantly stimulated by the crude root exudates of two duckweed species, which exhibited no effect on the growth of the P. fluorescens. The stimulation of the crude root exudates of HZ1 (HO) was significantly higher than that of WX3 (WO), which was consistent with the earlier research that the nitrogen removal abilities of HZ1 was higher than WX3 in treating the polluted water. An effect of different fractions of two duckweed root exudates was also observed. Among these fractions, only the acidic fraction of root exudates of WX3 (WA) inhibited the nitrogen removal efficiency of P. fluorescens, while all the fractions of root exudates of HZ1 displayed a stimulatory effect or no effect. Overall, the nitrogen removal efficiency of P. fluorescens firstly increased and then decreased with doses of duckweed root exudates and their fractions increase, with the exception of WA, where the efficiency remained unchanged after the initial decrease. When the dose was 1.0% (v/v), all of the fractions had the maximum effect. Therefore, these results indicated a close relationship between the root exudates and the nitrogen removal abilities of coupled duckweed-microbe systems, and the acidic faction was mainly responsible for the different effects of root exudates from two duckweed species on the nitrogen removal efficiency of P. fluorescens.