The development of methods for converting nitrogen oxides in water into valuable resources such as ammonia and hydrazine has been given some attention. By utilizing the nitrite-reducing catalytic activity of nitrite reductase (NiR), nitrite in water can be converted into ammonium. However, there are few reports in the research that synthesized ammonium from nitrite using nitrite reductase. Therefore, we aimed to investigate the effect of temperature on the nitrite-reducing catalytic activity of NiR from spinach in the presence of one-electron reduced riboflavin by kinetic analysis to find the optimum temperature conditions. The results of this study showed that the reaction temperature does not need to be higher than 296.15 K in order to improve the efficiency of ammonium production from nitrite using NiR.