Microbial immobilization of nitrate (NO3−) is considered to be an important process in reducing NO3 − accumulation and regulating nitrogen (N) retention capacity in soils. Accurately quantifying the rate of microbial NO3− immobilization is, therefore, necessary to predict its role in lowering NO3− accumulation in soils. We compared microbial NO3− immobilization rates using a 15NO3− labelling technique in three different ways: (a) 15N pool dilution, (b) organic 15N recovery and (c) microbial biomass 15N recovery, in a nitrate‐rich upland soil with and without amendment with organic materials with differing carbon‐to‐nitrogen ratios (C/N). The three methods generated similar NO3− immobilization rates, except when the soil was amended with easily decomposable organic materials (glucose and sucrose). We also developed a microbial NO3− immobilization‐specific quality index that incorporates the C/N ratio, lignin, cellulose and hemicellulose contents and pH for slowly decomposing organic materials (plant residues). This study provides direct empirical evidence that the results of different methods for calculating soil microbial NO3− immobilization rates are affected by the characteristics of organic materials added to the soil. Highlights Three methods for estimating microbial NO3− immobilization were compared The methods generated similar NO3− immobilization rates when amended with plant residues Higher NO3− immobilization when measured with 15N dilution with readily available C input A microbial NO3− immobilization‐specific index for plant residues was developed