Frequent changes in the tide levels in estuaries cause constant changes in the hydraulics of fish passage systems, with important effects on successful fish passage and swimming behavior. In most cases, Japanese eels often have low passage rates in engineered fishways because of their special habits. In this study, we established a 1:4 scaled-down weir-hole combination bulkhead fishway, studied the effects of different tidal differences and water depths on the passage rates and swimming behavior of yellow-phase Japanese eels, and analyzed the response of the Japanese eels to the hydraulic factors by superimposing their swimming trajectories and the flow field simulation results. We found that the passage rate of the eels decreased from 68.18% to 50.00% and 45.45% under extreme high tide differences and extreme low tide differences, respectively. The eels tended to use the low-velocity area to climb up the wall, and when crossing the mainstream, the yellow-phase Japanese eels preferred the area with a flow velocity of 0.1~0.36 m/s and a turbulent kinetic energy range of 0.001~0.007 m2/s2. Their upstream swimming speed was maintained at a range of 0.1~0.3 m/s.