This study developed a rapid, non-destructive method for the quantitative detection of protein in cottonseed by integrating near-infrared (NIR) fiber spectroscopy with chemometric machine learning. The establishment of this method holds significant importance for the rational and efficient utilization of cottonseed resources, advancing research on the genetic improvement of cottonseed nutritional quality, and promoting the development of equipment for raw cottonseed protein detection. Fuzzy cottonseed samples from three varieties were collected, and their NIR fiber-optic spectra were acquired. Reference protein contents were measured using the Kjeldahl method. Spectra were denoised through preprocessing, after which informative wavelengths were selected by combining Uninformative Variable Elimination (UVE) with Competitive Adaptive Reweighted Sampling (CARS) and the Random Frog (RF) algorithm. Partial least squares regression (PLSR), least-squares support vector machine (LSSVM), and support vector regression (SVR) models were then constructed to predict protein content. Model performance was assessed using the coefficient of determination (R²), root-mean-square error (RMSE), residual predictive deviation (RPD), and range error ratio (RER). The results indicate that the standard normal variate (SNV) is the most effective preprocessing step. The best performance was achieved by the LSSVM model coupled with UVE + CARS, yielding R² = 0.8571, RMSE = 0.0033, RPD = 2.7078, and RER = 10.72, outperforming the PLSR and SVR counterparts. These findings provide technical support for the rapid detection of fuzzy cottonseed protein and lay the groundwork for the development of related detection equipment.