Paddy N₂ fixation plays an important role for soil fertility maintenance, however, the relationship between rice planting and activity of diazotrophs to fix N₂ remains elusive. In this paper the amount of biological nitrogen fixation (BNF) and its distribution in different soil layers (0-1, 1-5, 5-15 cm) and rice were measured by ¹⁵N₂ direct labeling method. The abundances of nitrogen fixing bacteria and active nitrogen fixing bacteria in soil were determined by real-time quantitative PCR (qPCR). The results showed that rice planting significantly increased nitrogen fixation amount in each soil layer, and also increased the contribution of 1-5 cm and 5-15 cm layers to total nitrogen fixation. Total nitrogen fixation amount in rice planting treatment was 10.3 times higher than CK. The fixed N₂ allocated to rice plants accounted for 31.48% of the total nitrogen fixation. In 0-1cm soil layer, rice planting significantly increased the number of nifH RNA gene, but did not significantly increase the number of nifH DNA gene. These results indicate that rice planting did not increase the number of nitrogen fixing bacteria. The increase of nitrogen fixation amount in rice field is because rice planting greatly promoted the expression of nifH gene of nitrogen fixing bacteria, thus improved nitrogen fixation activity of nitrogen fixing bacteria.