Proline-directed (S/T-P) phosphorylation constitutes a fundamental yet incompletely understood neural signaling mechanism implicated in cerebral pathophysiology, including ischemic stroke. Building on our prior identification of a regulatory domain controlling the proline-directed kinase activity of GSK3β, we develop Nb.29E9, a nanobody that selectively inhibits S/T-P phosphorylation of key substrates, including RBM38, HIF1α, and p53. Under oxygen-glucose deprivation/reoxygenation (OGD/R) conditions, Nb.29E9 enhances neuronal and microglial viability, reduces reactive oxygen species (ROS), and mitigates neuroinflammation. Phosphoproteomic profiling reveals that Nb.29E9 broadly reshapes S/T-P phosphorylation networks in neural cells. To enable CNS delivery, we engineer MMP9-responsive TPNbT-CHFn nanoparticle with dual neuron/microglia-targeting capacity that effectively penetrate the blood-brain barrier. In a murine middle cerebral artery occlusion/reperfusion (MCAO/R) model, nanoparticle-mediated delivery of Nb.29E9 significantly reduces infarct volume, restores neurovascular homeostasis, and improves motor function. Mechanistically, Nb.29E9 normalizes pathological hyperphosphorylation of SMAD2/3-Thr8 (TGFβ signaling), CAMKK2-Ser495 (AMPK pathway), and AKT1S1-Ser183 (mTORC1 regulation). This study establishes GSK3β-driven S/T-P phosphorylation as a critical driver of ischemic neurodegeneration and presents a targeted nanotherapy platform with both mechanistic insight and therapeutic potential. Ischemic stroke is a leading cause of death and disability, yet therapeutic options remain limited. The progression from ischemic penumbra to ischemic core is governed by dynamic signaling events, particularly protein phosphorylation. Among these, glycogen synthase kinase 3β (GSK3β) plays a central role in neuronal injury, but current inhibitors lack activity-selectivity and brain delivery efficiency. Here, we identify proline-directed (S/T-P) phosphorylation mediated by GSK3β as a pathogenic driver of ischemic injury. Using interface-specific nanobody screening, we developed Nb.29E9, which binds the eIF4E2-GSK3β complex and selectively blocks S/T-P kinase activity without interfering with primed-substrate phosphorylation. Nb.29E9 protected neurons and microglia by suppressing apoptosis, reducing oxidative stress, and reprogramming inflammatory responses. To enable brain delivery, we engineered a ferritin-based nanoparticle (TPNbT-CHFn NP) with dual targeting and stroke-responsive release. Systemic administration of TPNbT-CHFn NP in murine MCAO/R models markedly reduced infarct volume, preserved neurovascular integrity, and improved motor recovery. This work uncovers pathogenic GSK3β signaling dynamics and establishes nanobody-based precision modulation combined with engineered delivery as a promising therapeutic strategy for ischemic stroke.