Confusing low-molecular-weight hyaluronic acid (LMWHA) from acid degradation and enzymatic hydrolysis (named LMWHA–A and LMWHA–E, respectively) will lead to health hazards and commercial risks. The purpose of this work is to analyze the structural differences between LMWHA–A and LMWHA–E, and then achieve a fast and accurate classification based on near-infrared (NIR) spectroscopy and machine learning. First, we combined nuclear magnetic resonance (NMR), Fourier transform infrared (FTIR) spectroscopy, two-dimensional correlated NIR spectroscopy (2DCOS), and aquaphotomics to analyze the structural differences between LMWHA–A and LMWHA–E. Second, we compared the dimensionality reduction methods including principal component analysis (PCA), kernel PCA (KPCA), and t-distributed stochastic neighbor embedding (t-SNE). Finally, the differences in classification effect of traditional machine learning methods including partial least squares–discriminant analysis (PLS-DA), support vector classification (SVC), and random forest (RF) as well as deep learning methods including one-dimensional convolutional neural network (1D-CNN) and long short-term memory (LSTM) were compared. The results showed that genetic algorithm (GA)–SVC and RF were the best performers in traditional machine learning, but their highest accuracy in the test dataset was 90%, while the accuracy of 1D-CNN and LSTM models in the training dataset and test dataset classification was 100%. The results of this study show that compared with traditional machine learning, the deep learning models were better for the classification of LMWHA–A and LMWHA–E. Our research provides a new methodological reference for the rapid and accurate classification of biological macromolecules.