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Objectives: Biologic disease-modifying antirheumatic drugs (bDMARDs) have been frequently used to treat juvenile idiopathic arthritis (JIA) resistant to classical (c) DMARDs. There is concern that vaccines may reduce vaccine effectiveness due to their immunosuppressive effects. This study aimed to evaluate the prevalence of anti-hepatitis B surface (HBs) antibody positivity in JIA patients treated with bDMARDs and to compare it with that in JIA patients treated with cDMARDs and in healthy controls. Materials and Methods: Anti-HBs antibody positivity and titers were compared between patients with JIA treated with bDMARDs or only cDMARDs, followed in our clinic, and healthy controls aged 2-20 years. All participants were vaccinated in infancy according to the routine vaccination schedule in our country. Anti-HBs titers ≥10 IU/L were considered seroprotective. Results: Ninety-two JIA patients receiving cDMARDs, 92 receiving bDMARDs, and 91 healthy controls were included in the study. The median time from the last vaccination to the study was 12.5 (min-max:3.5-17.55) years in controls, 12.5 (min-max:1.75-19.5) years in patients with cDMARDs, and 13.0 (min-max:2.25-19.0) years in patients with bDMARDs (p=0.060). Anti-HBs was positive in 50 (54.9%) controls, 66 (71.7%) patients with cDMARDs, and 62 (67.4%) patients with bDMARDs (p=0.048). Age, time from vaccination to study, and duration of bDMARD use were found to be risk factors for anti-HBs negativity in univariate regression analyses. According to the multivariable analysis of these three variables, the duration of bDMARD use was an independent risk factor for anti-HBs negativity (OR: 1.023, 95% CI: 1.005-1.041, p=0.013). Anti-HBs negativity was associated with a duration of bDMARDs longer than 32 months (AUC: 0.658, 95%CI: 0.541-0.776, p=0.014) with 60.0% sensitivity, and 59.7% specificity. Conclusions: A longer duration of bDMARDs was found to be a risk factor for anti-HBs negativity. Physicians should be careful in terms of anti-HBs negativity when the duration of biologics use is prolonged.