Background: Blood transfusion is an intervention used to save life particularly for those patients who survive only with receiving blood. Establishing effective diagnostic test menus concerning the screening of transfusion-transmissible infections in the blood banks play a vital role to safeguard recipients from transfusion-transmissible infections. Objective: The aim of this study was to assess blood transfusion-transmissible malaria and its screening cost analysis in Hawassa regional blood bank, Hawassa, Sothern Ethiopia. Methods: An institutional-based cross-sectional study was conducted from April to May 2018 among 414 voluntary blood donors. Each participant’s blood sample was screened for most transfusion-transmissible infections using antigen/antibody tests, while rapid diagnostic test and microscopy were used for malaria screening and confirmation. In addition, the cost screening of transfusion-transmissible infections was calculated using activity-based costing method. Results: The overall seropositivity of transfusion-transmissible infections was 7.0% and the positivity rate of hepatitis B virus, syphilis, and Plasmodium falciparum was 5.6%, 1.0%, and 0.5%, respectively. The cost per test of each transfusion-transmissible infection was US$5.04 for human immunodeficiency virus, US$4.61 for hepatitis B virus, US$5.11 for hepatitis C virus, and US$4.75 for syphilis, while the cost per test of malaria rapid diagnostic test was US$4.74 and this is comparatively lower than the cost per test of other transfusion-transmissible infections except for hepatitis B virus. In addition, total cost of laboratory incurred for transfusion-transmissible infections screening is estimated to be US$213,634.5 per year, while it becomes US$265,537.5 if the malaria screening cost is added. This means 19.54% of the total cost of laboratory incurred per year or US$51,903. Conclusion: The positivity rate of malaria parasites among voluntary blood donors was 0.5%, and it might be increased if the study was conducted in high transmission seasons. A cost of malaria screening is comparatively lower than costs of other transfusion-transmissible infections except for hepatitis B virus. Therefore, the screening of malaria parasites should be considered as one of the test menus of transfusion-transmissible infections in blood banks, especially in malaria-endemic areas.