Nitrogen fixation is an important process in marine nitrogen recycling. It can enhance marine primary productivity and marine carbon fixation capacity and reduce CO₂ concentration in the atmosphere, thus mitigating the greenhouse effect. The Beibu Gulf is a semi-enclosed bay located northwest of the South China Sea. In the Beibu Gulf, P and Fe are influenced by river input and atmospheric deposition, and the water in Beibu Gulf is rich in colloids. In order to study the effects of P, Fe, and colloids on the nitrogen fixation rate and nitrogen fixation in response to the nutritional environment, we conducted a field addition experiment for P, Fe, and colloid simulation in the spring of 2007. Surface water samples were collected from nine study sites between April and May 2007. P, Fe, and colloids were separately added for a scene simulation in the study sites of C02, C05, C07, and C09. Concentration gradients of P (potassium dihydrogen phosphate) were set as C, C+0.08, C+0.16, C+0.32, and C+0.48 μmol/L. Concentration gradients of Fe were set as C, C+0.22, C+0.44, C+0.88, and C+1.32 μmol/L (C is the concentration of nutrients in in situ water samples). Concentration gradients of colloids (particle size, between 0.22 μm and 10 kDa) were set as 0%, 2%, 50%, and 100%. The acetylene-reducing activity method was used to study nitrogen fixation and factors that affect it. The results showed that the contributions of temperature and salinity to the nitrogen fixation rate were low, while P, Fe, and colloids increased the nitrogen fixation rate. The nitrogen fixation rate was greatly promoted by adding colloids, while the addition of P and Fe did not affect the nitrogen fixation rate to a great extent. However, variations in the nitrogen fixation rate due to the addition of P, Fe, and colloids were -97% to 545%, -86% to 146%, and -96% to 1456%, respectively, and variations in chlorophyll contents were -24% to 50%, -32% to 36%, -53% to 41%, respectively. Differences in the nitrogen fixation rate were due to different nutrient concentrations in the surface water of the study sites in the spring of the Beibu Gulf. The nitrogen fixation rate was strongly related to dissolved nitrogen/dissolved phosphorus (DP), which indicated that a large amount of DP was consumed by organisms that perform nitrogen fixation. The high nutrient content in the Beibu Gulf may be the reason why the nitrogen fixation rate in the Beibu Gulf was lower than that in the oligotrophic sea. Moreover, there was no significant correlation between the rate of phytoplankton growth and nitrogen fixation rate. Phytoplankton growth limited the amount of nitrogen, and nitrogen fixation limited the amount of phosphorus. Effects of nitrogen fixation, which was limited by nutrients in the Beibu Gulf, could have a great influence on nitrogen sources and nitrogen biogeochemical processes. The results suggest that nitrogen may be a limiting factor for marine primary productivity in the spring in the Beibu Gulf, phosphorus is a limiting factor for the nitrogen fixation rate, and the addition of colloids enhances the nitrogen fixation rate.