Both straw addition and water management affect biological nitrogen fixation (BNF), but it is still unclear how straw addition impacts BNF under continuous or intermittent flooding irrigation in a rice cropping system. A 15N2-labelling chamber system placed in a rice field was used to evaluate BNF under straw addition with continuous or intermittent flooding irrigation for 90 days. The nitrogenase reductase (nifH) gene and reverse transcription gene (cDNA) in the soil were amplified using Real-time polymerase chain reaction (qPCR), and nifH gene high-throughput sequencing was applied. The BNF in the straw addition treatment (14.0 g straw added to 1.40 kg soil) under continuous flooding (14.3 kg ha-1) was 116% higher than that under intermittent flooding (6.62 kg ha-1). Straw addition and continuous flooding showed significant synergistic effects on BNF and the gene copy numbers of nifH DNA and nifH cDNA. The increase in BNF was mainly due to the increase in the abundance of heterotrophic diazotrophs such as Desulfovibrio, Azonexus, and Azotobacter. These results indicate that straw addition under continuous flooding stimulates BNF relative to intermittent flooding, which may ultimately lead to a rapid enhancement of soil fertility.