Introduction: Early diagnosis of ischaemic stroke and prompt implementation of causal treatment can improve patient outcomes. Recently, special attention has been paid to biomarkers associated with neuronal damage. One of them is neuronspecific enolase (NSE). Studies conducted to date indicate an increase in NSE concentration in patients with acute ischaemic stroke. The aim of this study was to assess serum NSE concentrations in patients with acute ischaemic stroke, and to determine the possible relationship between NSE levels and clinical features of the disease. Materials and methods: This case-control study included 60 patients with acute ischaemic stroke and 60 healthy controls (Opole, Poland, 2023). Demographic and clinical data were collected in the study group, and laboratory tests were performed. NSE concentrations in blood serum were determined in both groups. Results: No statistically significant differences were observed in the mean NSE concentrations between the study and control groups. However, above-normal NSE values were observed significantly more frequently in patients with ischaemic stroke. Significant differences in NSE levels were noted depending on the type of stroke (highest for partial anterior circulation infarct, PACI), stroke aetiology (highest for atherogenic stroke), and the level of disability (highest in completely dependent patients). Above-normal NSE values were also significantly associated with a longer time since the occurrence of ischaemic stroke. Conclusions: The type and aetiology of stroke, time from the first symptoms and degree of dependence influenced the serum concentration of NSE in stroke patients. Further research in this area is warranted.