Improving water use efficiency (WUE) is important in the water-deficient region in the North China Plain (NCP). How to assess the WUE of different management practices is becoming more important. The aim of the current study was to evaluate the relationship between instantaneous WUE, time-integrated WUE and carbon isotope discrimination (_13C) in winter wheat (Triticum aestivum L.) using different water and nitrogen (N) supplies. Three water levels and five nitrogen (N) levels with 15 treatments were set up in a pot experiment using PVC tubes with 1 m depth and 20 cm diameter buried in a field for two seasons. Biomass, root weight, grain yield, evapotranspiration (ET), gas exchange parameters and kernel _13C were measured to estimate the plant and leaf WUE. The results showed that the water regime and N supply strongly affected the aboveground and belowground biomass and WUE. The WUE was significantly improved when the N supply was increased from deprivation to higher conditions. When soil evaporation was prevented, the yield of winter wheat was linearly related to ET. The water supply had positive effects, while the N supply had negative effects on the gas exchange parameters. The impact of the interactions between the water regime and N supply on gas exchange parameters was significant, but an interactive effect of the two parameters on final crop yield and WUE was not observed. While both the water regime and the N supply had significant effects on the kernel _13C, the water regime had a greater effect on _13C than did the N supply. This study supported the use of the kernel _13C to evaluate crop WUE. However, the relationship between the instantaneous WUE calculated from the gas exchange parameters and the final WUE at yield level was not always consistent. A strong positive relationship between these two factors existed during the grain-fill stage when different management practices implemented their time-integrated effects on the crop growth. No such relationship was found during other growing stages. This study verifies that gas exchange WUE values at the grain-filling stage might provide a tool to assess the final WUE of different management practices.