Abstract: 【Objectives】Biogas slurry is one of the byproducts of breeding industry, full of organic and inorganic nutrients good for crop growth, and must be recycled from point of ecological and economic sustainability. The effects of biogas slurry on the soil fertility and biological properties of paddy soil, and the suitable use amount for healthy soil ecosystem were studied in this paper. 【Methods】Field experiments were carried out in early rice and late rice in Boluo County, Guangdong Province in 2013. The experiment included two controls(no fertilizer and chemical fertilizer), and five levels of biogas slurry treatments(200, 400, 600, 800 and 1000 m3/hm2). The soil nutrient contents and enzyme activities in paddy soil were analyzed, and the soil microbial diversities and microbial structures were studied by PCR-DGGE method. 【Results】 With biogas slurry application increased, the content of soil organic matter, available N,available P and available K were enhanced, continuous application of biogas slurry showed accumulation effects. Soil pH was reduced in early rice experiment and enhanced in late rice experiment with the increase of biogas slurry application. The activities of soil sucrase, urease and acid phosphatase were enhanced by the application. The highest soil microbial diversities indexes were obtained when the biogas slurry applied amount was 800 m3/hm2 in early rice and 600 m3/hm2 in late rice. The similarity coefficient of soil microbial structure was decreased with biogas slurry dosage increased; the differentiation of microbial structure among treatments in late rice season was higher than in early rice season. Principal component analysis showed that the first principal component in early rice was the information of physicochemical properties and microbial abundance, while that in late rice was the soil physicochemical properties, soil microbial diversities and enzyme activities except urease. Biogas slurry application affected paddy soil physicochemical properties, especially biological activity after continuous use in early and late rice. 【Conclusion】Application of biogas slurry increases the contents of soil organic matter, available N, P and K, and improves soil enzyme activities, microbial diversities and microbial structures in application range of biogas slurry 400-800 m3/hm2. Comprehensively considering the soil fertility and biological health, as well as the accumulation effects of continuous application, 600-800 m3/hm2 of biogas slurry is suggested for rice production in paddy soil ecosystem.