Anthocyanins are severity indicators for apple mosaic disease and can be used to monitor tree health. However, most of the current studies have focused on healthy leaves, and few studies have estimated the anthocyanin content in diseased leaves. In this study, we obtained the hyperspectral data of apple leaves with mosaic disease, analyzed the spectral characteristics of leaves with different degrees of Mosaic disease, constructed and screened the spectral index sensitive to anthocyanin content, and improved the estimation model. To improve the conciseness of the model, we integrated Variable Importance in Projection (VIP), Partial Least Squares Regression (PLSR), and Akaike Information Criterion (AIC) to select the optimal PLSR model and its independent variables. Sparrow Search Algorithm-Random Forest (SSA-RF) was used to improve accuracy. Results showed the following: (1) anthocyanin content increased gradually with the aggravation of disease. The reflectance of the blade spectrum in the visible band increased, the red edge moved to short wave, and the phenomenon of “blue shift of spectrum” occurred. (2) The VIP-PLSR-AIC selected 17 independent variables from 21 spectral indices. (3) Variables were used to construct PLSR, Back Propagation (BP), Support Vector Machine (SVM), Random Forest (RF), and SSA-RF to estimate anthocyanin content. Results showed the estimation accuracy and stability of the SSA-RF model were better than other models. The model set determination coefficient (R2) was up to 0.955, which is 0.047 higher than that of the RF model and 0.138 higher than that of the SVM model with the lowest accuracy. The model was constructed at the leaf scale and can provide a reference for other scale studies, including a theoretical basis for large-area, high-efficiency, high-precision anthocyanin estimation and monitoring of apple mosaics using remote sensing technology.