The rice-fish integrated system can reduce the application of chemical fertilizers and pesticides, ensure the normal growth of rice, maintain the environment quality of paddy field, and improve the economic benefits of farmers. In this study, “giant rice” and “Daohua carp” were selected as the experimental materials for the rice-fish integrated farming, and a two-year field experiment was carried out. Three farming modes were set up: rice-fish integrated farming with long-term flooding (URF30), rice planting alone with long-term flooding (UR30), and rice planting alone with dry-wet alternating (CK). Soil nutrient content, microbial biomass, and enzyme activities were analyzed at different rice growth stages, and the effects of rice-fish integrated farming on soil nutrients and enzyme activities were investigated. Results showed that the soil organic carbon (SOC) and total nitrogen (TN) contents of the URF30 treatment increased in two consecutive years, and were higher than those of other treatments. The grain yield of the URF30 treatment was also higher than other treatments. After short-term flooding for the UR30 and URF30 treatments, the soil nitrate and available phosphorus contents decreased, and the activities of soil β-glucosidase and urease significantly decreased. Long-term flooding in paddy fields decreased soil microbial biomass nitrogen (MBN) content. Compared to that at the beginning of flooding, soil MBN content in the UR30 treatment decreased significantly by 146.9% and 55.8% in 2020 and 2021, respectively. Fish farming in paddy field was beneficial to increase soil enzyme activities. In 2020 and 2021, the geometric mean of soil enzymes in the URF30 treatment were 5.8% and 7.5% higher than those of the UR30 treatment, and 19.4% and 15.6% higher than those of the CK treatment. The biomass of soil microorganisms were limited by soil phosphorus in the jointing stage of rice by short-term flooding, while were limited by nitrogen in the mature stage of rice by long-term flooding. The integrated farming of giant rice and fish could effectively guarantee soil fertility and soil enzyme activities under the condition of long-term flooding, and improve rice yield.