Ebolavirus (EBOV) is responsible for several EBOV disease (EVD) outbreaks in Africa, with a fatality rate of up to 90%. During 2014-2016, An epidemic of EVD spread throughout Sierra Leone, Guinea and Liberia, and killed over 11,000 people. EBOV began to circulate again in the Democratic Republic of Congo in 2018. Due to the need for a BSL-4 facility to manipulate this virus, the development and improvement of specific therapeutics has been hindered. As a result, it is imperative to perform reliable research on EBOV under lowered BSL restrictions. In this study, we developed a safe neutralization assay based on pseudotyped EBOV, which incorporates the glycoprotein of the 2014 EBOV epidemic strain into a lentivirus vector. Our results demonstrated that the tropism of pseudotyped EBOV was similar to that of authentic EBOV, but with only one infection cycle. And neutralizing activity of both authentic EBOV and pseudotyped EBOV were compared in neutralization assay using three different samples of antibody-based reagents against EBOV, similar results were obtained. In addition, an indirect ELISA was performed to show the relationship between IgG and neutralizing antibody against EBOV detected by our pseudotyped EBOV-based neutralization assay. As expected, the neutralizing antibody titers varied with the IgG titers detected by indirect ELISA, and a correlation between the results of the two assays was identified. By comparison with two different assays, the reliability of the results detected by the pseudotyped EBOV-based neutralization assay was confirmed. Collectively, in the absence of BSL-4 restrictions, pseudotyped EBOV production and neutralizing activity evaluation can be performed safely and in a manner that is neither labor- nor time-consuming, providing a simple and safe method for EBOV-neutralizing antibody detection and the assessment of immunogenicity of EBOV vaccines. All these remarkable advantages of the newly established assay highlight its potential to further application in assessment of immunogenicity of EBOV vaccine candidates.