Abstract: In recent years, due to human activities, a large amount of active nitrogen (N) has been transported into estuaries and coasts, causing eutrophication and harmful algal blooms. Understanding the distribution of different forms of N in estuarine systems and the relative preference of phytoplankton for different N forms is of great practical significance for the algal bloom mechanism exploration and the effective excess N management. In December 2022, environmental parameters and different forms of nitrogen concentrations were investigated in the Dongzhai estuary, Hainan Province. By using the ¹⁵N labelling technique, incubation experiments were carried out to quantify the phytoplankton uptake rates of Urea-N, ammonium (NH₄⁺), nitrate (NO₃⁻) and nitrite (NO₂⁻). The results showed that dissolved N concentrations in the water of Dongzhai estuary decreased along the river to the offshore in winter. The order of N concentration was NO₃⁻ > NH₄⁺ > NO₂⁻ > Urea-N, which ranged from 5.84−49.78 µmol/L, 1.16−40.64 µmol/L, 0.83−2.98 µmol/L and 0.25−0.98 µmol/L, respectively. The order of N uptake rates was NH₄⁺ > Urea-N > NO₃⁻ > NO₂⁻, which ranged from 46.6−280.1 nmol/L/h, 4.7−21.3 nmol/L/h, 1.1−9.8 nmol/L/h, and 0.03−3.4 nmol/L/h, respectively. Phytoplankton preferred reducing N (NH₄⁺ and urea-N), more specifically, the value of relative preference index (RPI) of NH₄⁺ RPI(NH₄⁺) and urea-N RPI(Urea-N) is 2.11−7.95 and 1.92−4.34, respectively, higher than the relative preference of oxidizing N (NO₃⁻ and NO₂⁻) both RPI(NO₃⁻) and RPI(NO₂⁻) were < 1. At the sampling stations with low and medium salinity, the preference for Urea-N sometimes exceeded that for NH₄⁺, i.e., RPI(Urea-N)/RPI(NH₄⁺) > 1. The study suggests that NO₃⁻ is the main dissolved N forms in Dongzhai estuary in winter, but phytoplankton prefers to utilize reduced N. Moreover, phytoplankton living in the upstream may have a higher preference for Urea-N than NH₄⁺. Our study enriches the understanding of different forms of N uptake by phytoplankton in the small estuary.