The mechanism underling dysfunction of cellular proteostasis on α-synuclein (αSyn) leading to pathogenesis of synucleinopathy remains unclear. Recently, we reported that the binding of an RNA secondary structure G-quadruplex (G4RNA) to a prion-like protein causes its sol-gel phase transition, leading to neurodegeneration in a hereditary neurodegenerative disease (Sci Adv. 2021). Herein, we demonstrate that G4RNA assembly forms scaffolds for αSyn aggregation, contributing to neurodegeneration. Purified αSyn binds to G4RNA specifically, but not other structures. G4RNA undergo Ca²⁺-induced phase separation and assembly, accelerating αSyn sol-gel phase transition. In mouse primary neurons, G4RNA aggregation was immediately observed under cellular stress conditions, thereafter co-aggregation of αSyn with G4RNA was occurred. Moreover, RIP-seq revealed endogenous G4RNA associated with αSyn aggregation. We also demonstrated that artificial assembly of G4RNA using an optogenetic approach initiated αSyn aggregation, thereby elicits neuronal dysfunction. Finally, G4 ligand can ameliorate G4RNA-elicited αSyn phase transition and neurodegeneration. These results suggest that G4RNA assembly evoked by various cellular stress triggers to develop aggregation of αSyn, which may be a cellular mechanism underlying onset of sporadic synucleinopathy.