This study utilized hyperspectral imaging technology combined with mathematical modeling methods to predict the protein content of rice grains. Firstly, the Kjeldahl method was used to determine the protein content of rice grains, and different preprocessing techniques were applied to the spectral information. Then, a prediction model for rice grain protein content was developed by combining the spectral data with the protein content. After performing multiplicative scatter correction (MSC) preprocessing and selecting feature wavelengths based on successive projections algorithm (SPA), the multivariate linear regression (MLR) model showed the best prediction performance, with a calibration set R2C of 0.9393, a validation set R2V of 0.8998, an RMSEV of 0.1725, and an RPD of 3.16. Finally, the quantitative protein content model was mapped pixel by pixel to visualize the distribution of rice protein, providing possibilities for non-destructive protein content detection.