Accurate quantification of forest biomass (FB) is the key to assessing the carbon budget of terrestrial ecosystems. Using remote sensing to apply inversion techniques to the estimation of FBs has recently become a research trend. However, the limitations of vertical scale analysis methods and the nonlinear distribution of forest biomass stratification have led to significant uncertainties in FB estimation. In this study, the biomass characteristics of forest vertical stratification were considered, and based on the integration of random forest and least squares (RF-LS) models, the FB prediction potential improved. The results indicated that compared with traditional biomass estimation methods, the overall R² of FB retrieval increased by 12.01%, and the root mean square error (RMSE) decreased by 7.50 Mg·hm⁻². The RF-LS model we established exhibited better performance in FB inversion and simulation assessments. The indicators of forest canopy height, soil organic matter content, and red-edge chlorophyll vegetation index had greater impacts on FB estimation. These indexes could be the focus of consideration in FB estimation using the integrated RF-LS model. Overall, this study provided an optimization method to map and evaluate FB by fine stratification of above-ground forest and reveals important indicators for FB inversion and the applicability of the RF-LS model. The results could be used as a reference for the accurate inversion of subtropical forest biomass parameters and estimation of carbon storage.