Abstract: The aggregation location of fish is more difficult to predict, due to the uncertain upstream path in the river. The proficient guidance of fish upstream behavior is essential to enhance the efficiency of fish passage facility inlets. In this research, a fish guide weir was introduced and assessed using three-dimensional numerical simulations of the flow field and the Active Fish Migration (AFM) model. An empirical test was carried out with the live fish within the context of the fish passage facilities in the Yaojiaping Water Conservancy Hub Project at the Hydropower Station. The results showed that the water surface differential between upstream and downstream of the fish guide weir ranged from 0.36 to 0.40 m. The flow velocity over the weir reached 1.5 to 2.8 m/s, indicating the barrier for fish migration. The singular low-flow velocity upstream channel was formed on the downstream side of the guide weir, thus directing fish towards the inlet of the fish collecting channel. The fish guide weir was then validated to select the exclusive route of the fish collecting channel. A fish migration model was used to predict the fish guide performance of the fish guide weir. The results demonstrated that there were over 90% of the fish trajectories in consistent patterns under both high and low downstream water level conditions. Specifically, the fish also migrated upstream along the right bank of the river and the low-flow channel on the downstream side of the fish guide weir. Ultimately, they congregated at the fish inlet of the fish collecting channel. In the fish release test, all tested individuals entered the fish collecting channel, further confirming that the fish guide weir effectively guided the fish swimming paths. A singular aggregation area was established at the fish inlet of the fish collecting channel. The fish guide weir can enrich the form of ecological hydraulic buildings. The finding can also provide a strong reference for the innovative hydraulic design at the inlet of the over-fish facility, particularly for the local improvement of the downstream river.