The effects of water depth, coverage rate and harvest regime on nutrient removal from wastewater and high-protein biomass production were assessed in a duckweed-based (Lemna aequinoctialis) pilot-scale wastewater treatment system (10 basins × 12 m(2)) that is located near Dianchi Lake in China. The results indicated that a water depth of 50 cm, a coverage rate of 150% and a harvest regime of 4 days were preferable conditions, under which excellent records of high-protein duckweed (dry matter production of 6.65 g/m(2)/d with crude protein content of 36.16% and phosphorus content of 1.46%) were obtained at a temperature of 12-21 °C. At the same time, the system achieved a removal efficiency of 66.16, 23.1, 48.3 and 76.52% for NH4(+)-N, TN, TP and turbidity, respectively, with the considerable removal rate of 0.465 g/m(2)/d for TN and 0.134 g/m(2)/d for TP at a hydraulic retention time of 6 days. In additionally, it was found that a lower duckweed density could lead to higher dissolved oxygen in the water and then a higher removal percentage of NH4(+)-N by nitrobacteria. This study obtains the preferable operation conditions for wastewater treatment and high-protein biomass production in a duckweed-based pilot-scale system, supplying an important reference for further large-scale applications of duckweed.