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In this study, immobilized microbial beads were proposed as a solution for excessive nitrogen concentration of the river sediment. The predominant denitrifying microbes were screened from the river sediment. The optimized production of immobilized microbial beads and long-term nitrogen removal efficiency were investigated. 16S rRNA gene sequencing analysis showed that denitrifying bacteria such as <i>Pseudomonas</i>, <i>Alcaligenes</i>, <i>Proteiniclasticum</i>, <i>Achromobacter</i> and <i>Methylobacillus</i> were dominant microflora in the enriched microbial agent, which accounted for 94.43% of the total microbes. <i>Pseudomonas</i> belongs to Gammaproteo bacteria, accounting for 49.22% and functioned as the most predominant denitrifying bacteria. The material concentration of 8% polyvinyl alcohol, 0.5% sodium alginate and 12.5% microbial biomass were found to be the optimal immobilizing conditions. The NH₄⁺-N and total nitrogen (TN) removal rates in sediment with dosing immobilized microbial beads were estimated as 68.1% and 67.8%, respectively, when compared to the dosing liquid microbial agent were 50.5% and 49.3%. Meanwhile, the NH₄⁺-N and TN removal rates in overlying water went up from 53.14% to 59.69% and from 68.03% to 78.13%, respectively, by using immobilized microbial beads.