Pot experiment in greenhouse with different application rates of bio-organic fertilizer on three kind soils were carried out for three years. Effects of fertilization on soil microbial biomass, enzyme activities and nutrients were studied. The results showed that, soil nutrients, microbial biomass and enzyme activity of treatments applied bio-organic fertilizer during three years increased compared with the no fertilization treatment in different contents of organic matter soils. The amounts of soil nutrients, microbial biomass and urease activity increased with the increase of application rate of bio-organic fertilizer, while pH value on the contrary. The soil invertase, polyphenol oxidase, protease activity increased first and then decreased, reached the highest when bio-organic fertilizer 10-30 g/kg applied in three kinds of soils, there was no significant change in catalase activity. Cotton dry matter accumulation reached the highest with application 10-20, 20-30 and 40 g/kg of bio-organic fertilizer, respectively in soils with higher, middle, and low contents of organic matter. Soil microbial biomass carbon and nitrogen decreased first and then increased and soil enzyme activity changed quite differently with the extension of fertilizer years in three kinds of soils. Soil nutrient, urease, invertase, protease activity and microbial biomass increased higher with bio-organic fertilizer applied in soil with the lower background organic matter.