Since hepatitis B virus (HBV) infections may cause serious clinical symptoms and become chronic, the appropriate use and interpretation of laboratory tests in diagnosis is crucial. Implementation of serological tests for all HBV markers leads to high cost, labour force and time loss, so diagnostic algorithms are used for this purpose. The aims of this retrospective study were to detect the number of unnecessary test requests in HBV infection serology and financial burden as its consequence and to find out the reasons and ways of solutions. Three-years records of microbiology laboratory of Dumlupinar University Evliya Celebi Training and Research Hospital, Kutahya, Turkey, between 2011-2013 were analysed for the detection of unnecessary test requests and a total of 179.640 HBV serological test results were evaluated. Serological tests requested more than one on the same day and HBeAg, anti-HBe and anti-HBc IgM tests required from the patients with natural immunity or vaccinated ones were considered as unnecessary test requests. The main reasons of unnecessary test requests were detected as follows; unable to check the previous HBV test results in HIMS (Hospital Information Management System) owing to restricted of time, not being friendly user of HIMS, not trusting the test results, not knowing diagnostics algorithms, requiring of the tests by secretaries or other staff like nurses, not questioning HBV vaccination and requiring screening tests because of the defensive medicine. Survey questions prepared to search these reasons and awareness level of diagnostic algorithms were handed and asked to fill out 146 physicians working at clinics which made over 100 unnecessary requests in 3 years. Total numbers of unnecessary requested serological HBV tests were 5415 (3.01%) and total financial burden for our hospital in three years was 42.256 TL (18.373 USD) according to the notifications of Turkish Social Security Institution. It was determined that the most unnecessary test requests were HBeAg (44.86%), anti-HBe (37.75%) and anti-HBc IgM (37.41%). The most unnecessary test requests were made by internal medicine, obstetrics and gynaecology, general surgery and cardiology clinics, respectively. According to questionnaires filled by the 100 clinicians, the most common causes of unnecessary tests were hepatitis test requests from hospitalized patients for screening (96%), the tests required by secretary or nurse (62%), pre-surgical screening tests (53%) and lack of knowledge about diagnostic algorithms (42%). In conclusion, it is suggested that test requests should be restricted by using screening algorithms and in-service training should be provided, tests required by secretary or nurse should be stopped and previous test results should be conveyed to the clinicians actively through HIMS in order to prevent unnecessary test requests causing economic and workforce loss in HBV infection serology. These measures can provide early and true diagnosis for the patients and also will reduce the economic loss.