Field trials were conducted to evaluate the effects of rotation and bio-organic fertilizer application on soil physiochemical properties，enzyme activities and microbial communities of pineapple under continuous cropping.The results showed that，compared with pineapple continuous cropping，the pineapple yield of pineapple-sugarcane rotation and pineapple-sugarcane rotation with bio-organic fertilizer application treatments significantly increased by 18.56% and 39.07%；the soil pH value increased by 0.50 and 0.66，which significantly alleviated soil acidification；the contents of soil nitrate nitrogen，available phosphorus，available potassium，organic matter significantly increased by 28.49% and 57.67%，20.49% and 39.77%，20.78% and 45.23%，10.38% and 23.10%，respectively；the content of soil microbial biomass carbon significantly increased by 12.10% and 29.56%，and the content of soil microbial biomass nitrogen significantly increased by 17.99% and 61.45%，respectively，while the soil microbial biomass carbon / microbial biomass nitrogen value decreased.In addition，the quantity of soil bacteria and actinomycetes was significantly increased，while the quantity of fungi was significantly reduced，and the utilization of carbon source by soil microorganisms was enhanced，and the activities of soil sucrase，urease，acid phosphatase and catalase were increased in rotation and rotation with bio-organic fertilizer application treatments.Under the experimental conditions，rotation and bio-organic fertilizer application could weaken soil acidification，improve soil microbial community structure and enhance fertility，and the optimum results were observed in the rotation with bio-organic fertilizer treatment.