Hydroacoustics is a non-invasive fish stock assessment sampling technique that plays an important role in fishery science and management. However, non-standard hydroacoustic surveys could lead to biased results, and the factor of the sampling period (e.g., season and diel cycle) is extremely critical as it can greatly affect hydroacoustic results. Efforts to improve the accuracy and credibility of the hydroacoustic survey results are getting more and more attention. Thus, we conducted two diel hydroacoustic surveys in situ in summer and winter to detect whether there were diel and seasonal differences in density, Target Strength (TS) and vertical distribution of fish. The results indicated that nighttime had significantly higher fish mean density than daytime in summer and winter. No significant difference between summer and winter daytime, however, significant difference between summer and winter nighttime, but this bias could be accepted from the fisheries management perspective; The mean TS of the summer daytime was significantly higher than that of summer nighttime, winter daytime and winter nighttime, but there were no significant differences among summer nighttime, winter daytime, and winter nighttime, mainly due to mean TS may be overestimated from fish schooling behavior during summer daytime; The fish vertical distribution had significant seasonal correlations and was more dispersed in different water layers during the nighttime, proving that the assessment was better at nighttime than during the daytime. Consequently, the hydroacoustic surveys in Yudong Reservoir and other similar plateau deep water reservoirs should be performed at nighttime, which will yield relatively accurate density and TS, and dispersed vertical distribution of fish.