Assimilating Sentinel-2 images with the CERES-Wheat model can improve the precision of winter wheat yield estimates at a regional scale. To verify this method, we applied the ensemble Kalman filter (EnKF) to assimilate the leaf area index (LAI) derived from Sentinel-2 data and simulated by the CERES-Wheat model. From this, we obtained the assimilated daily LAI during the growth stage of winter wheat across three counties located in the southeast of the Loess Plateau in China: Xiangfen, Xinjiang, and Wenxi. We assigned LAI weights at different growth stages by comparing the improved analytic hierarchy method, the entropy method, and the normalized combination weighting method, and constructed a yield estimation model with the measurements to accurately estimate the yield of winter wheat. We found that the changes of assimilated LAI during the growth stage of winter wheat strongly agreed with the simulated LAI. With the correction of the derived LAI from the Sentinel-2 images, the LAI from the green-up stage to the heading–filling stage was enhanced, while the LAI decrease from the milking stage was slowed down, which was more in line with the actual changes of LAI for winter wheat. We also compared the simulated and derived LAI and found the assimilated LAI had reduced the root mean square error (RMSE) by 0.43 and 0.29 m2 m–2, respectively, based on the measured LAI. The assimilation improved the estimation accuracy of the LAI time series. The highest determination coefficient (R2) was 0.8627 and the lowest RMSE was 472.92 kg ha–1 in the regression of the yields estimated by the normalized weighted assimilated LAI method and measurements. The relative error of the estimated yield of winter wheat in the study counties was less than 1％, suggesting that Sentinel-2 data with high spatial-temporal resolution can be assimilated with the CERES-Wheat model to obtain more accurate regional yield estimates.