Objective   To explore the potential molecular mechanisms of pramipexole in the treatment of restless legs syndrome (RLS) and provide a basis for clinical therapy.  Methods   RLS and pramipexole-related genes were screened using DisGeNET, GeneCards, and NCBI databases to identify shared genes. Functional enrichment and protein-protein interaction (PPI) analyses were performed via the STRING database, and a hub gene network was constructed using Cytoscape. The expression of key genes was validated in the GEO datasets (GSE54839, GSE37171). Molecular docking and molecular dynamics simulations were used to assess the binding stability between pramipexole and key genes. Finally, the effects of pramipexole were evaluated in maintenance hemodialysis (MHD) patients with RLS based on clinical indicators.  Results  The bioinformatics analysis shows that SLC6A3 is significantly upregulated in the GEO datasets (GSE54839, GSE37171), with high classification performance (GSE54839: AUC =0.755; GSE37171: AUC =0.606). Molecular docking and molecular dynamics analysis revealed that SLC6A3 binds stably with pramipexole and exhibits strong structural stability under dynamic conditions. In the clinical cohort, the relative serum expression level of SLC6A3 in RLS patients is significantly higher compared to the MHD group [(2.29±0.388) vs. (1.30±0.422), t=4.603, P＜0.0001] and the control group [(2.29±0.388) vs. (1.30±0.422), t=4.603,  P＜0.0001]. It is negatively correlated with dopamine levels (R²=0.601, P＜0.001). Pramipexole treatment significantly improves the IRLS (International Restless Legs Syndrome Rating Scale) score in RLS patients [(15.8±1.67) vs. (21.8 ±4.31), t=4.379, P＜0.001], and modulates the expression levels of SLC6A3 [treatment group (1.64±0.41) vs. control group (2.31±0.39), t=4.112, P＜0.001] and dopamine [treatment group (231.50±13.03) vs.control group (195.50±14.09), t=3.461, P＜0.001].   Conclusion  SLC6A3 is a key therapeutic target of pramipexole for RLS and may serve as a potential biomarker, providing new insights for personalized treatment of RLS.