Rationales: Restless leg syndrome (RLS) is a common complication in patients with end-stage renal disease (ESRD). However, there is a lack of biomarkers linking uremic RLS to dopaminergic neurons. Previous studies demonstrated that Tc-99m TRODAT-1 SPECT was a biomarker for RLS but the correlation between the physiologic parameter was lacking. Methods: Overall, 32 patients were enrolled in the study and divided into the following 3 groups: (1) control (n = 13), (2) ESRD without RLS (n = 8) and (3) ESRD with RLS (n = 11). All patients had a clinical diagnosis of RLS and received Tc-99m TRODAT-1 SPECT. A subgroup analysis was performed to compare differences between the control and ESRD with RLS groups. Tc-99m TRODAT-1 SPECT was performed and activities in the striatum and occipital areas were measured using manually delineated regions of interest (ROIs) by an experienced nuclear medicine radiologist who was blinded to clinical data. Results: The total ratio of Tc-99m TRODAT SPECT was lower in the ESRD with RLS group (p = 0.046). The uptake ratio of TRODAT negatively correlated with serum parathyroid hormone (r = −0.577, p = 0.015) and ferritin (r = −0.464, p = 0.039) concentrations. However, the uptake positively correlated with the hemoglobin concentration (r = 0.531, p = 0.011). The sensitivity and specificity of the total TRODAT ratio for predicting RLS in the overall population were 95.0% and 67.7%, respectively, at a cutoff value of 0.980 (area under the curve of receiver operating characteristic curve was 0.767, p = 0.024). Conclusion: In patients with ESRD and RLS, Tc-99m TRODAT might be a potential biomarker. Dysregulated hemoglobin, serum parathyroid hormone and serum ferritin concentrations might influence the uptake of the TRODAT ratio.