Hepatitis B infection (HBV) disease is one of the main hazard factors for chronic hepatitis, liver fibrosis, cirrhosis and hepatocellular malignant growth (HCC), which is a significant worldwide medical issue Hepatitis B virus (HBV) infection is one of the leading risk factors for chronic hepatitis, liver fibrosis, cirrhosis and hepatocellular cancer (HCC), which is a major global health problemeven with vaccine use and self-resolution in most cases. HBV contains different particle forms including non-infectious spherical and tubular sub viral particles (SVPs) with 22-nm-diameter that present potent immunogenicity. The main goal of the presented study was to optimize the best route for creation the SVP which discharged from incorporated HepG2.2.15 cell line in lab. For aim achievement, incorporated HepG2.2.15 cell line refined for SVP creation and fixation. SVP analyzed by using serological marker and electron microscopy.  Outcomes demonstrated the development of HepG2.2.15 cell line in   our complete media (Williams E media) with heat inactivated fetal cow-like serum (FBS), insulin and hydrocortisone bring about creation high measures of discharged viral particles in the supernatant. For recognizable proof of morphology and structures of SVP, electron microscopy indicated various shapes including circular and fibers for having a similar morphology of HBV virion, while it's completely different in the diameter. These findings shed light on an important technique used in production of huge number of SVP, which is important advance in Hepatitis BV irus irresistible rounds, and the gathering of HBV subviral particles might be basically connected to  pathogenesis of the virus inorder to enhance overcome of HBV infection.