This study addressed if long-term combined application of organic manure and inorganic fertilizers could improve the synchrony between nitrogen (N) supply and crop demand. (15) N-labeled urea was applied to micro-plots within three different fertilized treatments (no fertilizer, No-F soil; inorganic NPK fertilizers, NPK soil; and manure plus inorganic NPK fertilizers, MNPK soil) of a long-term field trial (1990-2009) in a dryland wheat field in the south Loess Plateau, China. After one season of wheat harvest, (15) N use efficiency was 20, 58, and 65 % in the No-F, NPK, and MNPK soil, respectively. During the early wheat growth stage, microbial immobilization of applied (15) N was significantly (P < 0.05) highest in the MNPK soil (15.3 %), higher in the NPK soil (12.6 %), and lowest in the No-F soil (7.4 %). Of the (15) N immobilized by the soil microbial biomass, 69 % (NPK soil) to 83 % (MNPK soil) was released between the stem elongation and flowering of wheat. Compared with the NPK soil, the MNPK soil had significantly (P < 0.05) higher grain yield. Our findings highlight that long-term application of organic manure with inorganic fertilizers cannot only improve the synchrony of N supply for crop demand but also increase N use efficiency and grain yield.