Reliable estimations of parameter values and associated uncertainties are crucial for crop model applications in agro-environmental research. However, estimating many parameters simultaneously for different types of response variables is difficult. This becomes more complicated for grapevines with different phenotypes between varieties and training systems. Our study aims to evaluate how a standard least square approach can be used to calibrate a complex grapevine model for simulating both the phenology (flowering and harvest date) and yield of four different variety–training systems in the Douro Demarcated Region, northern Portugal. An objective function is defined to search for the best-fit parameters that result in the minimum value of the unweighted sum of the normalized Root Mean Squared Error (nRMSE) of the studied variables. Parameter uncertainties are estimated as how a given parameter value can determine the total prediction variability caused by variations in the other parameter combinations. The results indicate that the best-estimated parameters show a satisfactory predictive performance, with a mean bias of −2 to 4 days for phenology and −232 to 159 kg/ha for yield. The corresponding variance in the observed data was generally well reproduced, except for one occasion. These parameters are a good trade-off to achieve results close to the best possible fit of each response variable. No parameter combinations can achieve minimum errors simultaneously for phenology and yield, where the best fit to one variable can lead to a poor fit to another. The proposed parameter uncertainty analysis is particularly useful to select the best-fit parameter values when several choices with equal performance occur. A global sensitivity analysis is applied where the fruit-setting parameters are identified as key determinants for yield simulations. Overall, the approach (including uncertainty analysis) is relatively simple and straightforward without specific pre-conditions (e.g., model continuity), which can be easily applied for other models and crops. However, a challenge has been identified, which is associated with the appropriate assumption of the model errors, where a combination of various calibration approaches might be essential to have a more robust parameter estimation.