Abstract: Method for preparation of pure hepatitis B surface antigen HBsAg from human plasma, wherein the initial plasma after being treated to remove lipides and partially remove human plasma proteins, is submitted to digestion with pepsin in amount of 0.01-0.50 mg/mg of protein to selectively decompose the plasma proteins while the antigen remains intact, filtered in a phosphate buffered medium, up to a pH value of 7.1-7.3, through molecular filters passing molecules of up to 100,000 Daltons, the infectiosity being subsequently de-activated by means of formaldehyde, in proportion of 1:2000 by volume, for 96 hours.