The extensive application of nitrogen (N) fertilizers in agriculture has resulted in a considerable accumulation of N in the soil, particularly nitrate (NO3−), which can be easily lost to the surrounding environments through leaching and denitrification. Improving the immobilization of NO3− by soil microorganisms in agriculture is crucial to improve soil N retention capacity and reduce the risk of NO3− loss. In this paper, we reviewed the significance of microbial immobilization of soil NO3− in soil N retention, the techniques to quantify soil gross microbial NO3− immobilization rate, and its influencing factors. Specifically, we discussed the respective contribution of fungi and bacteria in soil NO3− retention, and we clarified that the incorporation of organic materials is of vital importance in enhancing soil microbial NO3− immobilization capacities in agricultural soils. However, there is still a lack of research on the utilization of NO3− by microorganisms of different functional groups in soil due to the limited techniques. In the future, attention should be paid to how to regulate the microbial NO3− immobilization to make soil NO3− supply capacity match better with the crop N demand, thereby improving N use efficiency and reducing NO3− losses.