The invention discloses a method for preparing lentiviral particle packaged EBOV RNA as a positive reference substance for EBOV nucleic acid detection. The preparation method comprises the following steps: connecting part of segments of three genes NP, GP and L of EBOV in series and then integrating the segments into an expression vector of lentivirus, separately amplifying the constructed lentiviral expression plasmid and packaging plasmid in an engineering escherichia coli strain to obtain a large number of plasmids, then jointly transfecting the constructed lentiviral expression plasmid andpackaging plasmid into HEK293T cells, and carrying out cell expression to obtain a large number of lentiviral particles packaged with RNA genes of EBOV. The invention relates to the technical field of gene engineering. According to the method for preparing lentiviral particle packaged EBOV RNA as a positive reference substance for EBOV nucleic acid detection. The obtained particles are used as apositive reference substance for EBOV nucleic acid detection and have the advantage of well simulating the whole process from virus nucleic acid extraction to nucleic acid detection in an actual sample, and freeze-dried powder obtained by freeze-drying pseudovirus particles of EBOV through a freeze-drying protective agent is very stable.