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To investigate the effects of different sources of organic matter on soil biochemical properties and crop productivity and to find the best substitute for cow manure in the fields, a field experiment was carried out in a wheat-maize cropping system during 2012-2015. Three types of fertilizer applications, including a mineral fertilizer (CK), straw (S) and biogas slurry (B) were compared with cow manure (M) under an equal dose of nitrogen. In the 0-20 cm soil layer, the soil total nitrogen, microbial biomass nitrogen and carbon content of the M treatment was the highest, and the total organic carbon equivalent ratio of M decreased by 28.60% respectively, relative to S. Compared with CK, S and B, the urease activity equivalent ratio in the M treatment increased by 52, 12 and 21%, and the invertase activity equivalent ratio increased by 21, 20 and 26%, respectively. There were no significant differences in the hydrogen peroxidase activity among the four treatments. The annual crop yield and water use efficiency of the M treatment was significantly higher than other treatments, followed by S, B and CK. Our findings indicated that straw returning was the best substitute for cow manure.