Based on Savitzky-Golay (SG) smoothing screening, principal component analysis (PCA) combined with separately supervised linear discriminant analysis (LDA) and unsupervised hierarchical clustering analysis (HCA) were used for non-destructive visible and near-infrared (Vis-NIR) detection for breed screening of transgenic sugarcane. A random and stability-dependent framework of calibration, prediction, and validation was proposed. A total of 456 samples of sugarcane leaves planting in the elongating stage were collected from the field, which was composed of 306 transgenic (positive) samples containing Bt and Bar gene and 150 non-transgenic (negative) samples. A total of 156 samples (negative 50 and positive 106) were randomly selected as the validation set; the remaining samples (negative 100 and positive 200, a total of 300 samples) were used as the modeling set, and then the modeling set was subdivided into calibration (negative 50 and positive 100, a total of 150 samples) and prediction sets (negative 50 and positive 100, a total of 150 samples) for 50 times. The number of SG smoothing points was expanded, while some modes of higher derivative were removed because of small absolute value, and a total of 264 smoothing modes were used for screening. The pairwise combinations of first three principal components were used, and then the optimal combination of principal components was selected according to the model effect. Based on all divisions of calibration and prediction sets and all SG smoothing modes, the SG-PCA-LDA and SG-PCA-HCA models were established, the model parameters were optimized based on the average prediction effect for all divisions to produce modeling stability. Finally, the model validation was performed by validation set. With SG smoothing, the modeling accuracy and stability of PCA-LDA, PCA-HCA were significantly improved. For the optimal SG-PCA-LDA model, the recognition rate of positive and negative validation samples were 94.3%, 96.0%; and were 92.5%, 98.0% for the optimal SG-PCA-LDA model, respectively. Conclusion: Vis-NIR spectroscopic pattern recognition combined with SG smoothing could be used for accurate recognition of transgenic sugarcane leaves, and provided a convenient screening method for transgenic sugarcane breeding.