Milk vetch (Astragalus sinicus L.) is a winter-growing plant that can enhance soil fertility and provide essential nutrients for subsequent season crops. The fertilizing capacity of milk vetch is closely related to its above-ground biomass. Compared to the manual measurement methods of milk vetch biomass, remote sensing-based estimation methods have the advantages of rapid, noninvasive, and large-scale measurement. However, few studies have been conducted on remote sensing-based estimation of milk vetch biomass. To address this shortcoming, this study proposes combining unmanned aerial vehicle (UAV)-based hyperspectral imagery and machine learning algorithms for accurate estimation of milk vetch biomass. Through the analysis of hyperspectral images and feature selection based on the Pearson correlation and principal component analysis, vegetation indices (VIs), including near-infrared reflectance (NIR), red-edge spectral transform index (RE), and difference vegetation index (DVI), are selected as estimation metrics of the model development process. Four machine learning methods, including random forest (RF), multiple linear regression (MLR), deep neural network (DNN), and support vector machine (SVM), are used to construct the biomass models. The results show that the RF estimation model exhibits the highest coefficient of determination (R2) of 0.950 and the lowest relative root-mean-squared error (RRMSE) of 14.86% among all the models. Notably, the DNN model demonstrates promising performance on the test set, with the R2 and RRMSE values slightly superior and inferior to those of the RF, respectively. The proposed method based on UAV imagery and machine learning can provide an accurate and reliable large-scale estimation of milk vetch biomass.