Abstract: This study focuses on the endemic fish species of the southwestern mountainous region, Schizothorax prenanti, and quantitatively decoded its swimming behavior patterns to find its preferred hydrodynamic conditions and established a relational link between water flow conditions and ecological behavior. A flume with a flow gradient was used to create non-uniform flow field conditions and to obtain video images of the upstream movement of the fish in an indoor test flume. Using the image recognition technology, we calculated the swimming kinetic indicators of tail-beat angle and tail-beat frequency during the whole process of upstreaming, and realized the coupling study of ecology and hydrodynamics on this basis. The present study showed that the Schizothorax prenanti preferred to adapt to the non-uniform flow field by changing the tail-beat angle and tail-beat frequency at the flow gradient during the upstream process. Under the present experimental conditions, the preferred tail-beat angle ranged from 25° to 35°, the preferred tail-beat frequency ranged from 2.5 to 3.5 times/s, and the preferred flow velocity ranged from 0.20 to 0.40 m/s. With the increase of water velocity, the tail-beat angle showed a gradual decrease. The preference of the fish is to accelerate the tailing sprint in the area where the flow velocity changes from large to small, and the preference is for the tail-beat mode where the tail-beat angle changes to “weak-strong-weak”. The coefficient of gliding velocity was introduced in the gliding stage to quantify the coupling relationship between the tail-beat angle, gliding distance and flow velocity, and to calculate the contribution of the gliding distance to the displacement in the negative direction of the flow to obtain the angle between the gliding direction and the negative direction of the flow. It is shown that the value range of the coefficient of gliding flow velocity is representative at 1.0—3.0, and the preference range of the floundering splittail for the angle between the gliding direction and the negative direction of water flow is 40°—60°. This paper uses a multi-indicator quantitative evaluation method to further meet the construction needs of overfishing facilities with complex flow fields as background conditions.