The invention relates to a treponema pallidum IgM antibody biotin avidin enzyme-linked immune detection kit and a preparation method thereof, which relates to the syphilis detection. The kit comprises a concentrated washing liquid bottle, a negative and positive control bottle, a biotin mark antihuman IgM specific fragment mum chain monoclonal antibody bottle, a horseradish peroxidase mark avidin bottle, a TMB coloured solution bottle A, a coloured solution bottle B, a reaction stopping solution bottle, a micropore plate coated with recombinant antigen. The treponema pallidum has infectious standard strain Nichol strain and treponema pallidum non-infectious standard strain Reiter strain, by using a proteomics method, the treponema pallidum proteomics can be separated through dimensional electrophoresis, protein with strong immunogenicity can be identified by different patient or infectious animal serum, and then an antigen marker can be searched. The antigen marker is used for syphilis confirmation test, so that diagnosis sensitivity and specificity can be increased, a window stage is shortened. The treponema pallidum antibody can be sensitively detected and enables quantitative measurement, and has the advantages of cheap price, simple operation and accurate result.