Objective: To explore the utility of serum neurofilament light chain (sNfL) as a biomarker for primary and secondary neuroaxonal injury after ischemic stroke (IS) and study its value for the prediction of clinical outcome. Methods: We used an ultrasensitive single-molecule array (Simoa) assay to measure sNfL levels in healthy controls (HC, N=30) and two independent cohorts of patients with IS: (1) with serial serum sampling at hospital arrival (N=196), at days 2, 3, and 7 (N=89), and up to 6 months post-stroke; (2) with standardized MRI at baseline and at 6 months post-stroke, and with cross-sectional serum sampling at 6 months (N=95). We determined the temporal profile of sNfL levels, their association with imaging markers of neuroaxonal injury, and with clinical outcome. Results: Patients with IS had higher sNfL levels compared with HC starting from admission until 6 months post-stroke. sNfL levels peaked at day 7 (211.2 pg/ml [104.7–442.6], median [IQR]) and correlated with infarct volumes (day 7: partial r=0.736, p=1.5x10-15). 6 months post-stroke patients with recurrent ischemic lesions on MRI (N=19) had higher sNfL compared to those without new lesions (N=76, p=0.002). sNfL levels 6 months post-stroke further correlated with a quantitative measure of secondary neurodegeneration obtained from DTI MRI (r=0.361, p=0.001). sNfL levels 7 days post-stroke independently predicted modified Rankin scale scores 3 months post-stroke (cumulative OR [95% confidence interval] = 2.35 [1.60-3.45]; p=1.24x10-05). Conclusions: sNfL holds promise as a biomarker for monitoring primary and secondary neuroaxonal injury after IS and for predicting functional outcome.