The aim of this study is to model the hydrodynamic processes of the Istanbul Strait with its stratified flow characteristics, and calibrate the most important parameters using local and global search algorithms. For that, two open boundary conditions are defined, which are in the northern and southern parts of the Strait. Observed bathymetric, hydrographic, meteorological, and water-level data are used to set up the Delft3D-FLOW model. First, the sensitivities of the model parameters on the numerical model outputs are assessed using Parameter EStimation Tool (PEST) toolbox. Then, the model is calibrated based on the objective functions, focusing on the flow rates of the upper and lower layers. The salinity and temperature profiles of the strait are only used for model validation. The results show that the calibrated model outputs of the Istanbul Strait are reliable and consistent with the in situ measurements. The sensitivity analysis reveals that the spatial low-pass filter coefficient, horizontal eddy viscosity, Prandtl−Schmidt number, slope in log−log spectrum, and Manning roughness coefficient are most sensitive parameters affecting the flow rate performance of the model. The agreement between observed salinity profiles and simulated model outputs is promising, whereas the match between observed and simulated temperature profiles is weak, showing that the model can be improved, particularly for simulating the mixing layer.