An indoor and a field experiment were conducted to study the water-stable large aggregates, soil organic carbon content, saturated hydraulic conductivity, hydrogen isotope distribution, physiological characteristics of wheat photosynthesis and water utilization under long-term application of organic fertilizer and chemical fertilizer, thus ascertaining soil moisture distribution and water use mechanism of long-term application of organic fertilizer. The results showed that the organic fertilizer significantly increased the proportion of soil macro-aggregate, soil organic carbon content, saturated hydraulic conductivity, and thus led to the high stability of soil structure. The abundance of hydrogen and oxygen decreased gradually with the deepening of the soil layer. In the jointing period, the long-term application of chemical fertilizer mainly used the water in 0~60 cm soil layer, and the yield of organic fertilizer mainly used the water of 0~20 cm soil layer, while the contribution rate of soil moisture of 0~20 cm soil layer was significantly higher than that of other soil layers, reaching more than 50%. In the heading period, the long-term application of chemical fertilizer and organic fertilizer mainly used the water in 0~20 cm soil layer, and the contribution rate of soil moisture in this soil layer was still more than 50%. In the filling period, the long-term application of chemical fertilizer mainly used the water in 0~20 cm soil layer. The long-term application of organic fertilizer mainly used the water in 20~40 cm soil layer, and its contribution rate reached 50.2%. With the advancement of wheat growth period, the soil water storage decreased gradually. Long-term application of organic fertilizer was more conducive to reducing the ineffective loss of soil water in the early stage of wheat growth, which improved the soil water storage, and then promoted the supply of water in the late stage of wheat growth. Compared with application of chemical fertilizer, long-term application of organic fertilizer was more conducive to improving the photosynthetic rate, stomatal conductivity and leaf water use efficiency of wheat in different growth periods. With the advancement of wheat growth period, its above-ground biomass gradually increased and above-ground biomass of organic fertilizer was higher than that of chemical fertilizer. Ultimately, long-term application of organic carbon content significantly improved wheat yield and water use efficiency. Corcorrelation analysis showed that the water supply of 40~60 cm and 80~100 cm soil layers at jointing stage, and 20~80 cm soil layer at filling stage of wheat were more conducive to improving wheat yield, while soil supply of 40~60 cm, 80~100 cm at jointing stage, 60~100 cm at heading stage, and 80~100 cm at filling stage of wheat were more favorable to improve the water use efficiency. It concluded that the long-term application of organic fertilizer improved the soil structure, enhanced the storage, conservation and utilization capacity of precipitation in the soil, improved the photosynthetic physiological characteristics of wheat, and promoted the increase of yield and water saving and efficiency.