Despite urea being the most widely used nitrogen fertilizer, many plants show reduced growth and protein content when urea is the nitrogen source. The underlying causes of nitrogen metabolism imbalance in urea supply remain unclear. In this study, we discovered that plastidic glutamine synthetase (GS2) can improve the physiological traits and protein production of duckweed cultivated with urea. By comparing the urea utilization processes between two duckweed strains, LpZH0104 and Lp9595, which exhibited marked differences under the same urea treatment, we found that GS2 significantly impacts nitrogen metabolism. Overexpressing the GS2 gene in an efficient genetic transformation system of duckweed enhanced nitrogen‑carbon metabolism, leading to a 10 % increase in biomass and a 24 % increase in protein content in the overexpression lines (OE lines). Protein productivity and PPPN (protein production per unit of applied fertilizer N) increased by 47 % and 32 %, respectively. Conversely, silencing the GS2 gene reduced these traits. Additionally, transforming GS2 gene into rice, tobacco, and Arabidopsis also enhanced their biomass and protein content under urea cultivation conditions. This study highlights GS2's role in enhancing nitrogen metabolism and physiological traits in plants cultivated with urea, offering valuable insights for improving biomass and protein production in crops using urea fertilizer.