During replication of hepatitis B virus (HBV), HBV infected cells secrete infectious viral particles and large quantities of subviral particles which are assembled from surface protein without a viral genome. For the current study, the cell line of HepG2.2.15 genetically integrated with the HBV surface region was cultured as an expression system for in vitro production of hepatitis B virus subviral particles (HBVsvp) at different time for commercial purposes. Recorded Results showed that culturing and incubation of  HepG2.2.15 cells over 7 days incubation period resulted in continuous secretion of HBsvp in the supernatant.  ELISA's quantitative measurement of hepatitis B surface antigen reported that the highest titer of virus was day seven. Quantitative detection of HBsAg by ELISA indicated that the highest virus titer was at day seven. According to their size, subviral spherical particles recognized by a diameter of 22 nm. In conclusion, in vitro cultivation and incubation of HepG2.2.15 for secretion of HBsAg may be used in studying HBV replication thus, opening a new era for various studies from basic virology, in vitro diagnosis, to drug development against HBV genotypes.