Oral fluid (OF) sample collection and stability for HBsAg detection are not fully established. This study aims to investigate the applicability of OF collectors and sample stability for Hepatitis B virus surface antigen detection. Methods: Paired serum and OF samples were obtained from 191 individuals, and Chembio (Chembio Diagnostic System, USA) and Salivette (Sarstedt, Germany) devices were used for OF collection. Two HBsAg enzyme immunoassays (EIAs) were used (HBsAg One kit, Radim, Rome, Italy and ETI-MAK-4, Dia- Sorin, Vercelli, Italy) to determine the most efficient method according OF collector. Sample volume, incubation time, and cutoff (CO) value were evaluated. The stability of OF samples was determined under different environmental conditions. Results: Chembio samples analyzed using DiaSorin EIA without modification of the manufacturer’s instructions, demonstrated a sensitivity of 95.24% and a specificity of 100%. Salivette samples analyzed with Radim EIA with receiver operating characteristic (ROC) curve for calculating the CO showed a sensitivity of 78.26% and a specificity of 89.88%. HBsAg was detected in Chembio and Salivette samples under different environmental conditions, but the Chembio samples were the most stable. Conclusions: Both collectors can be used for HBsAg detection in OF samples, but some modifications of commercial EIAs should be incorporated for Salivette device. OF samples were reliably stable and could be stored for up to 90 days at 2–8◦C..