With growth in the livestock and poultry farming industries, a large amount of animal excreta has been applied to agricultural soils as an organic fertilizer over the past 20 years. However, detrimental impacts of animal excreta applications on the quality of agricultural soils may occur, because the excreta can contain some harmful components, such as heavy metal and antibiotics. Soil quality, defined as "The capacity of soil to function", can reflect the physical, chemical and biological response of the soil to field management practices. The aim of this study was to determine the differences in soil quality after long-term application of organic manures to a rice-wheat rotation field established in 1988 on a Gley paddy soil. Treatments included poultry litter (PL), livestock manure (LM) and chemical fertilizer (CF). Soils from the cultivated horizon (Ap) and plow pan (P) were collected from the long-term experimental site in Jiangsu Province, China, and soil organic carbon, nutrient content, pore structure properties, aggregate stability indexes and microbial and biochemical properties were determined. Our study showed that (1) The PL and LM treatments significantly increased soil organic carbon content, total and available N, P and K, more than in the CF treatment and both in the cultivated horizon and plow pan (P<0.05). The Olsen-P content in the cultivated horizon receiving the PL and LM treatments increased by 7-8 times compared with the soil receiving the chemical fertilizer application, and the soil available K doubled. (2) Soil physical properties also improved significantly with the long-term application of PL and LM. The PL and LM applications further increased the volumes of soil macropore and mesopore in the Ap horizon by 148%-170% and 135%-175% over those in the CF treatment, respectively, while the soil micropore volume in the PL and LM treatments was less. Compared with the CF treatment, PL and LM treatments decreased the tensile strength of the soil aggregates in the Ap and P layers by 33.7%-36.9% and 31.2%-34.0%, respectively. In addition, soil aggregate wet stability index, represented by normal mean weight diameter, was significantly greater in the PL and LM treatments than the CF in both Ap and P layers. (3) Long-term application of PL and LM significantly improved soil microbial and biochemical properties compared with CF. The soil microbial biomass C and N in the LM treatment was the highest among three treatments, being 2.1 and 1.5 times greater than in the CF treatment. Compared with the CF treatment, PL and LM applications increased the soil microbial quotient by 38.4%-54.8% and 20.1%-59.6%, respectively. Also, the soil basal respiration rate in the PL and LM treatment was 42-138 mg/g, 2.0-3.0 times greater than in the CF treatment. Application of organic manures also changed soil enzyme activities. The urease and intervase activities of the soils receiving the PL and LM treatments were 1.0-2.8 mg/g, and 17.3-33.3 mg/g, 3.5-6.7 and 1.6-2.1 times higher than those of the soil receiving the CF. This study suggests that the long-term application of poultry litter and livestock manure can improve the soil quality in a rice-wheat system, more so than using only chemical fertilizer. A higher organic matter content and a lower amount of harmful substances in the poultry litter and livestock manure may be the reason why it had such a positive impact on soil quality.