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Knee osteoarthritis (KOA), a progressive joint disease, is a leading source of chronic pain and disability, and its diagnosis mainly depends on medical imaging findings and clinical symptoms. This study aimed to explore an auxiliary diagnostic technology and clinical efficacy evaluation in KOA based on surface-enhanced Raman scattering (SERS). Three sequential experiments were performed: 1) preliminary observation of the therapeutic effects of icariin (ICA); 2) using serum SERS spectra obtained from rat models belonging to sham group, KOA group and icariin treatment group, respectively, to analyze the KOA-related expression profiles; 3) employing partial least squares (PLS) and support vector machines (SVM) algorithms to establish KOA diagnosis model. Pathological changes verified the efficacy of icariin in KOA. Raman peak assignment combined with spectral difference analysis reflected the biochemical changes associated with KOA, including amino acid, carbohydrates and collagen. ICA intervention significantly reversed these changes, although full recovery could not be achieved. Based on PLS-SVM approach, the sensitivity, specificity and accuracy of 100%, 98.33% and 98.89%, respectively, were obtained for screening KOA. This work proves that SERS has great potential to be used as an auxiliary diagnostic technology for KOA, and is also helpful for the exploration of novel KOA treatment agent., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)