© 2021 Elsevier LtdObjective: The dependence between gingival crevicular fluid (GCF) of Interleukin-34 (IL-34) level and Receptor activator of nuclear factor -kB ligand/ osteoprotegerin (RANKL/OPG) ratio in the severity of periodontitis might reveal an unknown pathway of diseases with bone destruction. There is no study about the evaluation of IL-34 levels together with GCF RANKL and OPG levels in periodontitis patients before and after non-surgical periodontal treatment (NSPT). The objectives of this research were to investigate changes in the levels and relative ratios of IL-34, OPG, and RANKL in the GCF of patients with periodontitis before and after NSPT. Materials and Methods: 20 healthy participants (CTRL), 20 patients with stage 3-grade B periodontitis and 20 with stage 3-grade C periodontitis were recruited. GCF and clinical periodontal recordings were investigated at the baseline and 6 weeks after NSPT. Enzyme-linked immunosorbent assay (ELISA) were used for quantifying of GCF IL-34, RANKL and OPG levels and their relative ratios were calculated. Results: Greater values for GCF IL-34 and RANKL levels were found in the both of periodontitis groups than in CTRL group at baseline, whereas GCF OPG levels were statistically lower at baseline (P < 0.05). GCF IL-34 and RANKL levels decreased in the 6th week after NSPT in the both periodontitis groups, while the concentration OPG levels statistically increased (P < 0.05). Significantly positive correlations among the IL-34 with RANKL, sampled-site clinical attachment level (CAL), and gingival index (GI), whereas negative correlation with OPG were reported (P < 0.05). Conclusions: GCF IL-34 levels was high in patients with periodontitis and decreased after NSPT and its levels showed positive correlations with RANKL/OPG ratio levels CAL and GI. Determining of IL-34 levels together with RANKL/OPG ratio in GCF may therefore be valuable in detecting high risk individuals with periodontitis patients.