Elevated CO2 levels (eCO2) pose challenges to wheat (Triticum aestivum L.) growth, potentially leading to a decline in quality and productivity. This study addresses the effects of two ambient CO2 concentrations (aCO2, daytime/nighttime = 410/450 ± 30 ppm and eCO2, 550/600 ± 30 ppm) and two nitrogen (N) supplements (without N supply-N0 and with 100 mg N supply as urea per kg soil-N100) on wheat (T. aestivum cv. Yunmai) growth, N accumulation, and soil microbial communities related to ammonia oxidization. The data showed that the N supply effectively mitigated the negative impacts of eCO2 on wheat growth by reducing intercellular CO2 concentrations while enhancing photosynthesis parameters. Notably, the N supply significantly increased N concentrations in wheat tissues and biomass production, thereby boosting N accumulation in seeds, shoots, and roots. eCO2 increased the agronomic efficiency of applied N (AEN) and the physiological efficiency of applied N (PEN) under N supply. Plant tissue N concentrations and accumulations are positively related to plant biomass production and soil NO3--N. Additionally, the N supply increased the richness and evenness of the soil microbial community, particularly Nitrososphaeraceae, Nitrosospira, and Nitrosomonas, which responded differently to N availability under both aCO2 and eCO2. These results underscore the importance and complexity of optimizing N supply and eCO2 for enhancing crop tissue N accumulation and yield production as well as activating nitrification-related microbial activities for soil inorganic N availability under future global environment change scenarios.