In order to provide a scientific basis for the promotion of soil fertility and the sustainable development of agriculture, a fixed site field experiment was conducted to investigate the changes of soil microbial biomass carbon, nitrogen and dissolved organic carbon after long-term (six years) straw return and urea application in a winter wheat-summer maize rotation system from Guanzhong Plain in Northwest China. The experiment was arranged in a split block design with two main treatments and three subplots, full maize straw return of previous crop (S+N) and no straw return (N) as main plots, and three chemical nitrogen treatments 0, 168 and 252 kg/hm² with four replicates as sub-plots. The results showed that with time soil MBC decreased from tillering stage to wintering stage, then increased until jointing stage and declined later. Soil DOC increased from tillering stage to jointing stage and decreased later with time, the dynamic change of soil MBN in the whole wheat growing season showed a decreased trend. Compared with no straw return treatments, straw return significantly increased the MBC and DOC content in the soil, and increased by 6.7% and 9.3% in average, respectively. MBN in straw return treatments were higher than no straw return treatments, but significantly increased only in wintering stage, jointing stage and maturity stage. With the increase of nitrogen rate, MBC and MBN significantly decreased. While with the increase of nitrogen rate, DOC significantly increased in straw return treatments, and increased by 11.8% in average, but the DOC in no straw return treatments showed an increased trend firstly and declined later. Overall, straw returning could obviously increase soil microbial biomass carbon, nitrogen and dissolved organic carbon, and the application of chemical nitrogen fertilizer promoted the decomposition of maize straw, but excessive application of chemical nitrogen fertilizer decreased the microbial biomass carbon and nitrogen, and will have negative effect on the growth of microorganism. So, straw returning with moderate nitrogen fertilizer is an effective measure to improve the soil fertility and realize the sustainable development of agriculture in the winter wheat-summer maize rotation system from Guanzhong Plain in Northwest China.