Application of livestock manure (LM) in field is an impartment way to solve the ecological environmental menace in the intensive and scale development of livestock farming industry. In this paper soils were sampled in five wheat-rice rotation fields on two soil types under long-term application of LM and chemical fertilizer (CF). Biochemical properties, such as soil microorganism composition, microbial biomass C and N (MBC, MBN), soil basal respiration (SBR), soil phosphatase, urease and invertase activities, were investigated as soil biochemical quality indicators. The results showed that, compared with CF treatment, the amounts of bacteria and actnomycete (+72%, +132%), the contents of MBC and MBN (+89%, +74%), the SBR and soil microbal quotient (+49%, +45%) increased significantly but the amount of fungi (-38%) significantly decreased in long-term application of LM treatment. The activities of soil urease and invertase were also higher in long-term application of LM than CF treatment. As greatly affected by pH values, soil microbial metabolic quotient (qCO2) and soil phosphatase changed indistinctly. Successive regression analysis indicated that the changes in soil active organic carbon content and physical and chemical properties are the main reasons for the changes in soil biochemical quality indicator in long-term application of LM.