Purpose: The aim of this study was to evaluate changes in levels of adrenergic receptor β3 (ADRB3), Rho-related kinase 2 (ROCK2), and guanine nucleotide exchange factor (GEF), which play key roles in the adrenergic and cholinergic pathways of contraction-relaxation harmony in voiding physiology, and to explore the relationship between these proteins and overactive bladder (OAB). Methods: This study included 60 idiopathic OAB patients and a healthy control group. A validated OAB-validated 8 questionnaire was completed by all participants. Serum levels of ADRB3, ROCK2, and GEF were examined with enzyme-linked immunosorbent assays. Patient and control groups were compared in terms of these levels, and receiver operating characteristic (ROC) curves were generated for all parameters. Results: The levels of ROCK2 were significantly elevated, but there were no correlations between the OAB symptom score and the serum levels of ROCK2, ADRB3, and GEF in OAB patients. In the ROC analysis, ROCK2 alone provided the strongest potential relationship (area under the curve=0.651) with 84.9% sensitivity. The ROCK2+GEF combination provided a satisfactory relationship (AUC=0.755). The AUC for the ADRB3+ROCK2+GEF combination was 0.752, with 64.2% sensitivity and 88.2% specificity. Conclusions The study results suggest that alterations in serum ROCK2 levels and the use of this parameter in combination with ADRB3 and GEF levels can shed light on the pathophysiology of idiopathic OAB syndrome and provide a new perspective for treatment.