Increasing evidence indicates that the hepatitis B virus (HBV) replicates in peripheral blood mononuclear cells (PBMCs), but at a low level. The present study aimed to establish a reliable and sensitive method that effectively detects HBV viral products for monitoring antiviral therapy, organ transplantation screening, and diagnosing occult HBV infection. In the present study, PBMCs (obtained from six healthy volunteers) were inoculated with HBV, and cultured with phytohemagglutinin (PHA) and interleukin‑2 (IL‑2) to stimulate cell proliferation. PBMCs were harvested, and quantitative detection of HBV DNA in cell suspension and intracellular hepatitis B surface antigen (HBsAg) was conducted on days 0, 1, 6 and 12, respectively. In situ hybridization, immunohistochemistry and reverse transcription‑polymerase chain reaction (RT‑PCR) were performed to analyze the HBV infection. The results demonstrated that HBV DNA increased concurrently with proliferation of PBMCs isolated from three of six healthy volunteers, and the mean number of PBMCs on day 12 was 13.61 times higher than the initially seeded cell number (P<0.01). The mean copies of HBV DNA at day 12 were 2.98 times higher compared with initial levels (P<0.05). Furthermore, intracellular HBsAg levels increased concurrently with proliferation of PBMCs in one group of cultured PBMCs, which was accompanied by increased HBV DNA levels. In addition, HBV nucleic acids were detected in PBMCs using in situ hybridization. Intracellular HBsAg was observed in PBMCs and HBV RNA was also detected by RT‑PCR. The present study demonstrated that HBV replicates in proliferating PBMCs, which were induced by PHA and IL‑2. This method offers a novel investigative tool to detect HBV infection in PBMCs and to monitor the course of HBV infection.