The second national pollution sources survey showed that the total nitrogen emission from aquaculture is 99100 tons in 2017. To protect the environment and human health, it is important to remove nitrogen from aquaculture wastewater before being discharged to surrounding waters. Biological denitrification is considered the most promising approach methods, since nitrate can be reduced to harmless nitrogen gas by bacteria. Sufficient carbon source is necessary during heterotrophic denitrification process. To solve the problems mentioned above, external carbon sources such as methanol, acetic acid and glucose were added to the wastewater, whereas they were generally high-cost, high-energy and high operating requirement. In contrast, agricultural wastes were used as carbon source, which has shown significant economic advantages and high-efficiency. Many aquaculture wastewater treatment systems often face variations in hydraulic retention time (HRT) and Influent nitrate concentration (INC) which are caused by acute change of wastewater characteristics and production, and HRT and INC often exert a profound effect on the treatment performance of biological treatment systems. The purpose of this study is to construct a solid-phase denitrification system with loofah sponge as carbon source, and investigate the effects of HRT and INC on the denitrification performance of loofah sponge-denitrification reactor (LS-DR), so as to provide a theoretical basis for the further optimization of denitrification process of loofah sponge as denitrification carbon source in aquaculture tailwater. Loofah sponge, one typical agricultural waste, was studied as the carbon source for solid phase denitrification under dynamic flow conditions by using 1-D column experiment. We aim to preliminarily investigate the LS-DR’s NO₃^--N, NO₂^--N, NH₄⁺-N, TN, TP and COD removal effect at different HRT (16, 20, 24 and 28 h) and INC (50, 75, 100 and 125 mg/L). The optimal HRT of denitrification reactor was optimized by one-way ANOVA analysis. And, the high-throughput sequencing technology based on Illumina MiSeq platform was used to analyze the bacterial community structure of LS-DR in the initial and final stages of operation. The results indicated that when INC=50 mg/L and HRT=24 h, the removal efficiency of both NO₃^--N and TN in LS-DR reached the highest value, which were 98.97%±0.52% and 97.84%±0.94% respectively. And NO₂^--N was also at a low level (< 0.5 mg/L). On the basis of HRT of 24 h, when INC increases to 75, 100 and 125 mg/L, the nitrate removal efficiency and nitrate removal rate (NRR) of LS-DR increased significantly with the increase of INC (P< 0.05), and the effluent COD decreased with the increase of INC, but LS-DR did not realize complete denitrification. It is worth noting that LS-DR can completely remove NH₄⁺-N throughout the experiment. After 14 days of operation, SEM results showed that the surface structure of LS was conducive to the attachment and growth of microorganisms; high throughput sequencing results showed that the dominant bacteria of LS-DR included Proteobacteria, Bacteroidetes, Campilobacterota, Firmicutes and Verrucomicrobiota. Among the identified bacteria, Thermomonas (1.46%), Thauera (0.55%), Azospira (3.32%), Simplicispira (1.01%), Pseudoxanthomonas (0.39%), Herbaspirillum (3.02%) and Uliginosibacterium (0.9%) can carry out denitrification. Cyphaga xylanolytica (1.61%) and Cloacibacterium (2.69%) are mainly involved in the degradation of towel gourd, Flavobacterium (1.17%) and Diaphorobacter (0.64%) can both denitrify and degrade LS. According to the analysis of the above results, it is considered that the optimal HRT of LS-DR is 24 h and the optimal INC is 50 mg/L. This study provides a reference for the optimization of loofah sponge solid-phase denitrification process and promotes the development and application of new slow-release carbon sources.