The aims of this study are to explore the effect of different scales in the high spectral data on the estimation of chlorophyll content of apple leaves, to find out the optimal differential window scale and to establish a model for estimating the chlorophyll content of apple leaves. Taking the apple leaves as the research object, the actual spectral reflectance of the leaves was determined by the ASD Field Spec 3 spectrometer and the chlorophyll contents of the leaves were measured in the laboratory. Firstly, the differential transformations from 1 to 30 window scales were done for actual spectral data respectively, and correlation analyses were done between apple leaf chlorophyll content and differential data, then two sensitive wavelengths were chosen under each window. Secondly, taking five consecutive differential windows as a group, the best differential window was selected in each group. Lastly, after the conversion of two sensitive wavelengths in six differential windows, relationship analyses between chlorophyll content of apple leaves and two sensitive wavelengths were done, then two new parameters with the largest correlation coefficient were chosen to establish estimation model. Results showed that with increasing differential window, the determination coefficient (R2) of estimation model decreased after an initial increase, the tipping point was at the 13th differential window scale. Testing the partial least squares (PLS) model and the stepwise regression (SR) model established under differential window scale of the 13th, the R2 of the SR model was higher than that of the PLS model. The RMSE and RE% of the SR model were lower than that of the PLS model, which showed that SR model was more suitable to estimate chlorophyll content.