Background <p>Serum Neurofilament light chain (sNfL) is a sensitive marker of axonal injury. This study investigated the relationships between carotid ultrasound findings, metabolic biomarkers, stroke severity, and functional outcome in patients with acute ischemic stroke (AIS). This case–control study included 130 patients with first-ever AIS and 40 age- and sex-matched healthy controls. All participants underwent NIH Stroke Scale (NIHSS) assessment, carotid duplex ultrasound, sNfL measurement, and laboratory testing within 24&#xa0;h of stroke onset. Associations between vascular/metabolic parameters, sNfL, stroke severity, and 3-month modified Rankin Scale (mRS) scores were analyzed.</p> Results <p>AIS patients had higher vascular comorbidities and higher sNfL levels than controls (307.82 ± 112.95 versus 43.55 ± 1.20 pg./mL, <i>p</i> &lt; 0.0001). Carotid intima–media thickness (IMT) was significantly greater in AIS, but without Doppler flow changes. Carotid IMT correlated positively with sNfL. Serum calcium and red blood cell count were inversely correlated with sNfL, while higher white blood cell count, bilirubin, INR, and lower prothrombin concentration were associated with baseline stroke severity. The white blood cell (WBC) count was associated with a poorer functional outcome (<i>p</i> = 0.040).</p> Conclusion <p>Carotid IMT and selected metabolic biomarkers were associated with sNfL, reflecting neuronal injury in AIS. While several laboratory parameters were associated with stroke severity, only a higher WBC count was associated with a poorer functional outcome.</p> Trial registration <p>this study was prospectively registered on the clinical trial website with registration ID NCT06090058. Available at <a href="https://clinicaltrials.gov/study/NCT06090058">https://clinicaltrials.gov/study/NCT06090058</a>.</p>