We conducted an experiment with five treatments in winter wheat of the dryland of Northwest China, i.e. 30 t·hm-2 cow dung (M) plus different doses of nitrogen fertilizer (0, 75, 150, 225, and 300 kg N·hm-2), denoted by M+N0, M+N75, M+N150, M+N225, and M+N300, respectively. After three years of treatment, wheat yield, grain quality, and soil biological characteristics were measured in two consecutive years (2018 and 2019). The results showed that the combination of manure with nitrogen fertilizer significantly increased wheat yield compared with the manure-only treatment (M+N0). Compared with the manure-only treatment, the combined treatments significantly increased wheat grain protein content, wet gluten, sedimentation value, and extensibility, but not for starch content. Neither wheat yield nor grain quality had significant differences among the M+N150, M+N225, and M+N300 treatments, but both were prominently higher than those of M+N75. Soil microbial biomass carbon (MBC) and nitrogen (MBN) reached highest in M+N150 for both years, which were distinctly higher than those of M+N0, M+N225, and M+N300. In 2018, soil β-1, 4-glucosidase, cellobiohydrolase, L-leucine aminopeptidase, β-1,4-N-acetyl glucosaminidase, and alkaline phosphatase activities in M+N150 treatment were higher than those of other treatments. In 2019, soil enzyme activities (excluding L-leucine aminopeptidase) in M+N150 were higher than those of M+N0 and M+N225. MBC significantly positively correlated with MBN, and both significantly positively correlated with the activities of cellobiohydrolase, β-1, 4-N-acetyl glucosaminidase, and alkaline phosphatase. MBN significantly positively correlated with total nitrogen content and negatively correlated with NO3-. Considering winter wheat yield, grain quality, and soil biological characteristics, M+N150 was conducive to sustainable production of winter wheat in drylands of Northwest China.