Objective This study aimed to compare the therapeutic efficacy of alteplase and urokinase in patients with acute ischemic stroke (AIS) and evaluate their effects on serum parameters. Methods In this retrospective study, 82 AIS patients admitted to the 950th hospital of PLA between October 2021 and October 2023 were enrolled and divided into two groups (n=41 each). The alteplase group received intravenous thrombolysis with alteplase (0.9 mg/kg), while the urokinase group received intravenous urokinase (1 million IU). Neurological deficits (assessed by NIHSS), functional outcomes (evaluated by mRS), hemodynamic parameters of middle cerebral artery, hemorheological indices and incidence of comlications were compared between the two groups before and after treatment. Results Following treatment, the alteplase group demonstrated significantly lower NIHSS and mRS scores compared to the urokinase group (P <0.001), showing greater improvements in both neurological deficit and functional status. Hemodynamic parameters, including peak systolic velocity, enddiastolic velocity, and mean flow velocity, showed greater improvement in the alteplase group (P<0.05). Similarly, hemorheological indices (plasma viscosity, whole blood high-shear viscosity, erythrocyte aggregation index, and hematocrit) improved more significantly in the alteplase group (P<0.001). Notably, complication rates were comparable between the two groups (P=0.532). Conclusion Alteplase demonstrates superior efficacy compared to urokinase in improving neurological function, cerebral hemodynamics, and hemorheological parameters in AIS patients, while maintaining a comparable safety profile. These findings support the use of alteplase as a preferred thrombolytic agent for AIS treatment.