Introduction: There are very limited data on the role of biomarkers correlating with the outcome in acute ischemic stroke (AIS). We evaluated the predictive values of the plasma concentrations of soluble serum stimulation-2 (sST2), matrix metalloproteinase-9 (MMP-9), and claudin-5 in AIS. Methods: The biomarker levels in the plasma samples of consecutive AIS patients collected at baseline, 12 h, and 24 h from stroke onset were quantified using immunoassays. Stroke severity was assessed using the National Institutes of Health Stroke Scale (NIHSS) and functional outcome at 90 days using the modified Rankin Scale (mRS), with scores above 3 defined as poor outcome. Receiver operating characteristic curve analysis and multiple logistic regression were performed for evaluating the discriminative power of each marker. Results: We included 108 patients in the study (mean age 62.3 ± 11.7 years). Median NIHSS score was 12 (interquartile range 8–18). High baseline glucose levels, systolic blood pressure, baseline NIHSS, low Alberta Stroke Program Early CT Score, and hemorrhagic transformation were associated with poor outcomes. Elevated sST2 at 12 h (50.4 ± 51.0 ng/mL; p = 0.047) and 24 h (81.8 ± 101.3 ng/mL; p = 0.001) positively correlated with poor outcomes. MMP-9 (p = 0.086) and claudin-5 (p = 0.2) were not significantly associated with the outcome, although increased expressions of both markers were observed at 12 h. Multiple logistic regression showed that sST2 levels ≥71.8 ng/mL at 24 h, with a specificity of 96.9%, emerged as an independent predictor of poor functional outcome (OR: 6.44; 95% CI: 1.40–46.3; p = 0.029). Conclusion: Evaluation of sST2 may act as a reliable biomarker of functional outcome in AIS.