Background: Carotid artery stenosis (CAS) is the major pathogen of cerebral infarction and brain death. Early detec-tion and risk prediction could help the diagnosis and improve the outcome of patients. The clinical significance of lncRNA GAS5 (GAS5) and miR-222-3p in the diagnosis and progno-sis of CAS was evaluated in this study to explore novel effec-tive biomarkers of CAS. Methods: A total of 72 CAS patients and 63 healthy indi-viduals (control) were enrolled in this study. The expres-sion levels of GAS5 and miR-222-3p in study subjects were detected using PCR. The ROC, Kaplan-Meier, and Cox regression analyses were carried out to estimate the diagnos-tic and prognostic value of GAS5 and miR-222-3p in CAS. The interaction between GAS5 and miR-222-3p was dis-closed by the dual-luciferase reporter. Results: The reduced expression of GAS5 and elevated expression of miR-222-3p were observed in CAS patients compared with the healthy controls, and a significant corre-lation between their expression levels in CAS was revealed. GAS5 and miR-222-3p could discriminate CAS patients from the healthy controls with high sensitivity and specificity. The GAS5 downregulation and miR-222-3p upregulation could predict the poor prognosis of CAS patients and may be asso-ciated with the severe development of patients. In human vascular smooth muscle cells, miR-222-3p could negatively regulate the luciferase activity of GAS5. Conclusion: Both GAS5 and miR-222-3p served as the diagnostic and prognostic biomarkers of CAS. The function of GAS5 might result from the regulation of miR-222-3p, which needs further validation.