A series of novel 4,5-bisindolyl-1,2,4-triazol-3-ones were designed, prepared and evaluated for their glycogen synthase kinase (GSK)-3β inhibitory activities. Compounds exhibited favorable inhibitory potency towards GSK-3β kinase at the molecular level and in cells indicated by significantly reducing GSK-3β substrate Tau phosphorylation at Ser396 in primary neurons showing the inhibition of cellular GSK-3β. In an in vitro model of neuronal injury, compounds 6b, 6d and 6f prevented glutamate-induced neuronal death which was closely associated with cerebral ischemic stroke. Preliminary structure-activity relationship was examined and showed that different substituents on the indole ring had significant influences on the GSK-3β inhibitory potency. These findings may provide new insights into the development of novel GSK-3β inhibitors as neuroprotective agents.