Lung cancer is the leading cause of death in cancer patients, and microwave ablation (MWA) has been extensively used in clinical treatment. In this study, we characterized the spectra of MWA-treated and untreated lung squamous cell carcinoma (LSCC) tissues, as well as healthy lung tissue, and conducted a preliminary analysis of spectral variations associated with MWA treatment. The results of characteristic spectral analysis of different types of tissues indicated that MWA treatment induces an increase in the content of nucleic acids, proteins, and lipid components in lung cancer tissues. The discriminant model based on the principal component analysis-linear discriminant analysis (PCA-LDA) algorithm together with leave-one-out cross validation (LOOCV) method yield the sensitivities of 90%, 80%, and 96%, and specificities of 86.2%, 93.8%, and 100% among untreated and MWA-treated cancerous tissue, and healthy lung tissue, respectively. These results indicate that Raman spectroscopy combined with multivariate analysis techniques can be used to explore the biochemical response mechanism of cancerous tissue to MWA therapy.