To understand the spatial distribution patterns and fish behaviour in the Xiaonanhai reach of the upper Yangtze River at the end of the Three Gorges Reservoir, four acoustic surveys were carried out over longer periods in 2011 and 2012, which included two boat surveys and two fixed-station horizontal detections. The results showed that fish assemblages were unevenly distributed in the investigated areas, and were mainly concentrated in the section from Funiuxi to Gaojiatuo. Fish behaviour was found to show a night-time activity peak. Additionally, fish moved much closer to the riverbank at night than during the day. Considering the intense human activities in this area, some suggestions are offered to mitigate the negative effects of shipping and hydrodam construction by using design and operational logistics to support future fish conservation.