To quantify the spatiotemporal distributions and biomass of fish resources in the Danjiangkou Reservoir, the investigations of the fish assemblage were conducted by the combination of hydroacoustic detection and multi-mesh gillnet sampling between June and September 2022, and between June and September 2023. During 2022 and 2023, a total of 53 fish species were collected, belonging to 5 orders and 10 families. In 2022, a total of 41 fish species were collected, among which Hemiculter leucisculu, Culter mongolicu, Squalidus argentatus and Pseudobrama simoni were dominant. In 2023, a total of 45 fish species were collected, among which Culter mongolicu and Hemiculter leucisculu were dominant. The average densities, sizes (target strengths) and biomasses of fish assemblage from hydroacoustic data showed obvious spatial and temporal heterogeneities. In terms of temporal distributions, the average fish density ((8779±143) ind./ha) and biomass ((60.57±1.21) kg/ha) in 2023 were significantly higher than those in 2022 ((3827±96) ind./ha and (37.76±0.95) kg/ha) (P＜0.01), while the average target strength in 2023 ((-56.25±0.16) dB) was significantly lower than that in 2022 (((-55.12±0.13) dB). In terms of horizontal distributions, the average densities, target strengths, and biomasses of fish assemblage in the Hubei region were consistently higher than those in the Henan region both in 2022 and 2023. In terms of vertical distributions, fish were mainly concentrated in the water layer above 30 m. The quantitative sampling data of gillnets were consistent with the results of hydroacoustics. In terms of temporal distributions, the average value of catch per unit effort of the whole reservoir in 2023 was higher than that in 2022, while the average value of fish total length was on the contrary. In terms of horizontal distributions, the average values of fish total length and catch per unit effort of the Hubei region were all higher than those of the Henan region both in 2022 and 2023. This study evaluated the characteristics of spatial-temporal distributions of fish resources in the Danjiangkou Reservoir for the first time, and the results could provide basic data support for water source protection, fishing ban effect assessment, and reservoir optimization operation.