The invention discloses a preparation method of human immunodeficiency virus (HIV) antibody detection test paper. The preparation method comprises the following steps of: spraying gold on a gold cushion by a mixed solution of a P74 recombinant antigen mark and a P44 recombinant antigen mark through membrane marking and gold spraying equipment to obtain a first gold cushion; spraying gold on the gold cushion by a rabbit IgG monoclonal antibody mark to obtain a second gold cushion; marking on a polyvinyl chloride bottom plate stuck with a nitrocellulose membrane by a detection line covering solution and a control line covering solution to obtain a polyvinyl chloride bottom plate with a marked membrane; assembling a sample cushion, the first gold cushion, the second gold cushion, the polyvinyl chloride bottom plate with the marked membrane and water absorption paper; and cutting the assembly into the detection test paper. In the manner, the detection test paper prepared by the preparation method disclosed by the invention can be used for judging whether an oral cavity sample contains an HIV antibody or not based on an immune lateral flow chromatography technology through manual operation and naked eye reading; the diagnosis is rapid, the result is accurate, and damages to a body of a subject are not caused.