Grasslands play a vital role in the global ecosystem. Efficient and reproducible methods for estimating the grassland aboveground biomass (AGB) are crucial for understanding grassland growth, promoting sustainable development, and assessing the carbon cycle. Currently, the available methods are limited by their computational inefficiency, model transfer, and sampling scale. Therefore, in this study, the estimation of grassland AGB over a large area was achieved by coupling the PROSAIL model with the support vector machine regression (SVR) method. The ill-posed inverse problem of the PROSAIL model was mitigated through kernel-based regularization using the SVR model. The Zoigê Plateau was used as the case study area, and the results demonstrated that the estimated biomass accurately reproduced the reference AGB map generated by zooming in on on-site measurements (R2 = 0.64, RMSE = 43.52 g/m2, RRMSE = 15.13%). The estimated AGB map also maintained a high fitting accuracy with field sampling data (R2 = 0.69, RMSE = 44.07 g/m2, RRMSE = 14.21%). Further, the generated time-series profiles of grass AGB for 2022 were consistent with the trends in local grass growth dynamics. The proposed method combines the advantages of the PROSAIL model and the regression algorithm, reduces the dependence on field sampling data, improves the universality and repeatability of grassland AGB estimation, and provides an efficient approach for grassland ecosystem construction and planning.