This study assesses the potential of using multispectral images collected by an unmanned aerial system (UAS) on machine learning (ML) frameworks to estimate cereal rye (Secale cereal L.) biomass. Multispectral images and ground-truth cereal rye biomass data were collected from 15 farmers’ fields up to three times between March and May in northwest Ohio. Images were processed to derive 13 vegetation indices (VIs). Out of 13 VIs, six optimal sets of VIs, including excess green (ExG), normalized green red difference index (NGRDI), soil adjusted vegetation index (SAVI), blue green ratio (B_G_ratio), red-edge triangular vegetation index (RTVI), and normalized difference red-edge (NDRE) were selected using the variance inflation factor (VIF) based feature selection approach. Six regression models including a multiple linear regression (MLR), elastic net (ENET), multivariate adaptive regression splines (MARS), support vector machine (SVM), random forest (RF), and extreme gradient boosting (XGB) were investigated for estimation of cereal rye biomass based on the VIs. For most of the models, the six selected VIs performed better than or similar to the full set of 13 VIs with R2 ranging from 0.24 to 0.59 and RMSE ranging from 83.13 to 91.89 g/m2 during 10-fold cross-validation. During independent accuracy assessment with the selected set of VIs, XGB exhibited the highest R2 (0.67) and lowest RMSE (83.13 g/m2) and MAE (48.13 g/m2) followed by RF and ENET. For all the models, the agreement between observed and predicted biomass was high for biomass less than or equal to 200 g/m2 but decreased for biomass greater than 200 g/m2. When field-collected structural features were integrated with the selected VIs, the models showed improved performance, with R2 and RMSE of the models reaching up to 0.82 and 61.67 g/m2 respectively. Among the six VIs, SAVI showed the strongest impact on the model prediction for the best-performing RF and XGB regression models. The findings of this study demonstrate the potential of precisely estimating and mapping cereal rye biomass based on UAS-captured multispectral images. Timely information on cover crop growth can facilitate numerous decision-making processes, including planning the planting operations, and management of nutrients, weeds, and soil moisture to improve agronomic and environmental outcomes.