Background: Early differentiation between acute ischemic stroke (AIS) and stroke mimics remains challenging in emergency setting. Molecular biomarkers such as matrix metalloproteinase-9 (MMP-9) may aid in improving diagnostic accuracy. Methods: A systematic literature search was conducted according to PRISMA guidelines across PubMed, EMBASE, Cochrane Library, and Web of Science. Studies assessing MMP-9 concentrations within 24 hours of symptom onset in AIS patients versus stroke mimics or healthy/matched controls ≥18 years were included. Random-effects meta-analyses were used to estimate pooled mean differences (MD) in MMP-9 levels between groups. Heterogeneity was assessed using I² and Cochran's Q. Results: Twenty-seven studies were included, with 16 (2,661 AIS patients, 1,283 all controls) meeting criteria for meta-analysis. Mean MMP-9 concentrations were significantly higher in AIS patients compared to all controls combined (MD: 93.9 ng/mL; 95%CI: 25.0-162.8; p=0.01, I²=96%, Cochran's Q: p<0.001). Subgroup analyses confirmed elevated levels in AIS patients versus healthy/matched controls (MD: 110.6 ng/mL; 95%CI:13.8-207.4; p=0.029) and versus stroke mimics (MD: 66.61 ng/mL; 95%CI: -6.2-139.4; p=0.01). Heterogeneity remained consistently high across comparisons (I² ≥ 95%; p<0.05). Conclusion: MMP-9 levels appear significantly elevated in AIS patients compared to both stroke mimics and healthy controls/matched controls, supporting its potential as a diagnostic biomarker in the acute setting. However, substantial inter-study heterogeneity limits generalizability, and standardized studies are needed to validate their clinical applicability. (PROSPERO: CRD42024611953) Funding: We acknowledge support from the German Research Foundation, the Medical Faculty Carl Gustav Carus, and the SLUB, as well as the Open Access Publication Funds of the TU Dresden. The funding bodies had no impact on any aspect of the study or the content of the manuscript.