The vertical slot fishway is a widely adopted fish passage structure in both domestic and international contexts, renowned for its efficient operation which is fundamentally dependent on superior hydraulic characteristics within the pool. In this study, we initially employed numerical simulation techniques to analyze and optimize the design. Guided by the principle that the mainstream should be centered within the pool with symmetrically distributed backflow areas, we selected three distinct layout configurations of the vertical slot fishway pool, each with varying degrees of mainstream sinuosity. Then, a physical model was constructed, utilizing grass carp (Ctenopharyngodon idella) as the target species for fish passage experiments. Comparative analyses were conducted to evaluate the upstream migration performance of grass carp across the three layout configurations. By integrating hydraulic performance metrics with the observed migration outcomes, we were able to recommend an optimal degree of mainstream sinuosity for the vertical slot fishway pool. The numerical simulation results revealed that as the degree of mainstream sinuosity increased, the scope of the mainstream expanded, the maximum attenuation of the mainstream along the pathway increased, and the velocity gradient across the vertical slot cross-section intensified. Conversely, both the mainstream velocity and the vertical slot velocity decreased. From the perspective of the target species, the experimental results demonstrated that in Case Ⅱ, grass carp exhibited a concentrated upstream migration trajectory, a continuous migration process, reduced tail flick frequency, shorter migration time, and a higher success rate. Therefore, Case Ⅱ is recommended as the optimal pool chamber layout in this study. This research primarily explores three layout configurations of the vertical slot fishway pool with varying degrees of mainstream sinuosity and identifies the optimal layout. The findings can provide valuable guidance for the design and structural optimization of vertical slot fishways in practical engineering applications.