Recently, the need for early diagnosis of modifiable risk factors involved in the etiology of stroke has been highlighted in the literature. Nesfatin-1 is a peptide expressed in the central nervous system and peripheral tissues and has been used as a biomarker in recent years. This study aimed to determine the association of ischemic stroke with internal carotid artery stenosis according to nesfatin-1 level and whether it could be used as a biomarker. A total of 118 patients were included in the study. Three groups were defined: acute stroke patients with symptomatic internal carotid artery stenosis, acute stroke patients without internal carotid artery stenosis, and a control group. Nesfatin-1 levels were measured and compared. The median value was 22 pg/mL in acute stroke patients with internal carotid artery stenosis, 24.3 pg/mL in acute stroke patients without internal carotid artery stenosis, and 46.4 pg/mL in the control group. There is a difference between the median values of nesfatin-1 according to the stroke groups with the control group ( < 0.001). When a cut-off value of ≤30.62 was taken for nesfatin-1, an AUC value of 0.773 indicated statistical significance ( < 0.001). Sensitivity was 77.03%, specificity 83.33%, PPV 90.48%, and NPV 63.83%. The main limitations of our study are the small sample size and the fact that the function of nesfatin-1 is not completely known. Although we found that nesfatin-1 levels were lower in ischemic stroke patients compared to controls, its diagnostic potential indicates a moderate discriminatory ability with an AUC value of 0.773. Therefore, whether it is suitable for clinical use will be demonstrated by studies in larger and multicenter cohorts.