Above-ground biomass (AGB) serves as an indicator of crop growth status, and acquiring timely AGB information is crucial for estimating crop yield and determining appropriate water and fertilizer inputs. Unmanned Aerial Vehicles (UAVs) equipped with RGB cameras offer an affordable and practical solution for efficiently obtaining crop AGB. However, traditional vegetation indices (VIs) alone are insufficient in capturing crop canopy structure, leading to poor estimation accuracy. Moreover, different flight heights and machine learning algorithms can impact estimation accuracy. Therefore, this study aims to enhance wheat AGB estimation accuracy by combining VIs, crop height, and texture features while investigating the influence of flight height and machine learning algorithms on estimation. During the heading and grain-filling stages of wheat, wheat AGB data and UAV RGB images were collected at flight heights of 30 m, 60 m, and 90 m. Machine learning algorithms, including Random Forest Regression (RFR), Gradient Boosting Regression Trees (GBRT), Ridge Regression (RR), Least Absolute Shrinkage and Selection Operator (Lasso) and Support Vector Regression (SVR), were utilized to construct wheat AGB estimation models. The research findings are as follows: (1) Estimation accuracy using VIs alone is relatively low, with R2 values ranging from 0.519 to 0.695. However, combining VIs with crop height and texture features improves estimation accuracy, with R2 values reaching 0.845 to 0.852. (2) Estimation accuracy gradually decreases with increasing flight height, resulting in R2 values of 0.519–0.852, 0.438–0.837, and 0.445–0.827 for flight heights of 30 m, 60 m, and 90 m, respectively. (3) The choice of machine learning algorithm significantly influences estimation accuracy, with RFR outperforming other machine learnings. In conclusion, UAV RGB images contain valuable crop canopy information, and effectively utilizing this information in conjunction with machine learning algorithms enables accurate wheat AGB estimation, providing a new approach for precision agriculture management using UAV remote sensing technology.