Objective: SARS-CoV-2 infection has been spread worldwide since 2019 and declared a pandemic infection. Unfortunately, humanity is still trying to deal with the infection. Under these circumstances, scientists head towards drug repurposing studies as the fastest solution for combatting SARS-CoV-2 viral infection. Betaferon (Interferon beta-1b) is a member of interferons, and its mechanism of action is the same as naturally produced interferon beta-1a in the immune system.Material and Method: In this study, the antiviral effect of Betaferon on SARS-CoV-2 infection in vitro and in silico was analyzed. The drug toxicity, gene expression, and docking calculations are evaluated. Result and Discussion: Betaferon showed significant antiviral activity against COVID-19. Furthermore, Betaferon decreased the expression of both viral entries mediating proteins such as ACE2 and TMRPSS2. Betaferon decreases not only the expression of TMPRSS2 but also the enzymatic activity of TMPRSS2. Furthermore, in silico analyses revealed that Betaferon interacts with viral Spike protein. Hence, a decrease in the expression of viral entry mediating proteins, inhibition of the activity of TMPRSS2, and interaction with viral Spike protein indicate that Betaferon has an antiviral activity for COVID-19 virus through inhibition of viral entry pathway.