This paper studied the effects of combined application of biogas slurry and chemical fertilizer under same N application rate on the quantities of bacteria, actinomycetes and fungi as well as the activities of urease, protease and catalase in winter wheat rhizosphere soil. With the growth of winter wheat, the quantities of test microorganisms and the activities of urease and catalase showed a trend of increasing after an initial decrease, while the protease activity showed an S-type change. Combined application of biogas slurry and chemical fertilizer increased the quantities of test microorganisms significantly, and improved the activities of soil urease and protease. Applying 50% biogas slurry N as basal plus 50% chemical N as topdressing and applying 25% biogas slurry N as basal plus 75% chemical N as topdressing had the best effect, while applying single conventional urea or biogas slurry had the worst effect. At all growth stages, the activity of soil catalase was the highest in treatments 25% biogas slurry N as basal plus 75% chemical N as topdressing and single biogas slurry, but had greater differences in other treatments among the growth stages. The results suggested that proper biogas slurry application combined with chemical fertilization could increase the microbial quantity and enzyme activities in winter wheat rhizosphere soil.