Abstract: In order to assess the current situation of fish resources in the Provincial Nature Reserve for Yangtze Cetacean of Zhenjiang, a simultaneous survey of fish resources was carried out in spring (April) and autumn (September) in 2020 by using hydroacoustic frequency difference technology and net survey method, and the temporal and spatial characteristics of fish resources in the surveyed river section were evaluated. Based on the analysis of fish echo image data of 200 and 333 kHz, the acoustic assessment of fish resources was carried out by frequency difference technique. The results of hydroacoustic survey showed that the average density of fish resources was (0.094±0.183) ind./m², and the average target strength (TS) of fish was (–57.33±5.69) dB, mainly distributed in the range of –62.5 to –50.5 dB. In addition, TS increases with increasing water depth. The results of net survey showed that 758 fishes (53.53 kg) were collected in the river section and a total of 48 species were identified, of which Cypriniformes dominated. The proportion of pelagic fish population quantity and mass was 36.15% and 66.12%, respectively. The proportion of the quantity and mass of fish (pelagic fish with body length less than 20 cm, body height less than 6 cm and body weight less than 100 g) fed on the Yangtze finless porpoise was 23.22% and 8.72%, respectively. In terms of time characteristics, there was no significant difference in fish resource density between April and September (P>0.05), and the average TS in April was smaller than that in September, and the average size of fish in April was smaller than that in September. In terms of spatial characteristics, the fish resources were mainly distributed in the waters near the north branch of He Changzhou and Jiaobei Beach, and the pelagic fish resources were relatively large, and the fish resources were more concentrated in April. Underwater acoustic frequency difference technology can effectively eliminate plankton signals and background noise signals in the water environment, which is of reference significance for fishery acoustic evaluation in the Yangtze River Basin. The results of this study can provide a scientific basis for the accurate assessment of the bait fish resources of the Yangtze finless porpoise in the reserve, support the conservation of the Yangtze finless porpoise in situ, and provide a scientific basis for the conservation of fish resources in the lower Reaches of the Yangtze River.