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Soybean polypeptide is a protein hydrolysate. It has multiple benefits, such as anticancer, antioxidant, etc. Addressing the limitations of traditional methods, such as time-consuming procedures and high costs, this study proposed a novel approach for the rapid, nondestructive detection of soybean polypeptide content. This approach utilizes the fusion of spectral and image information. First, seven preprocessing methods were applied to the spectral data. The results indicated that the model accuracy was highest after applying the standard normal variate preprocessing method. Second, a combined method, consisting of synergy interval partial least squares and iteratively retains informative variables, was proposed to extract characteristic variables. A two-layer Stacking framework ensemble learning model was constructed to compare the detection effects between single spectral input and fusion of spectral and image information input. The results showed that the fusion input model achieved the best performance. The coefficient of determination (R2) of prediction of the fusion input model reached 0.952, an improvement of 0.061 compared to single spectral input. And root mean square error of prediction (RMSEP) of the fusion input model reached 1.803 × 10−4, a reduction of 7.43 × 10−5 compared to single spectral input. These results indicate that the fusion of spectral and image information can effectively improve the model's accuracy, offering valuable technical support for soybean breeding and quality detection. [ABSTRACT FROM AUTHOR]