The invention discloses a test paper strip for detecting HIV antibody in urine, a detection cup and a preparation method thereof. The test paper strip includes a bottom plate, a sample pad, a nitrocellulose membrane and water-absorbing paper arranged on the bottom plate. The two ends of the nitrocellulose membrane are overlapped with the sample pad and absorbent paper respectively, and the mouse anti-human IgG antibody labeled with colloidal gold and the gp160 antigen labeled with colloidal gold are sprayed on the sample pad near the end of the nitrocellulose membrane. , the nitrocellulose membrane is provided with the first detection line coated with HIV recombinant antigen gp41 and HIV recombinant antigen gp160, and the control line coated with goat anti-mouse antibody; the test strip of the present invention is added with new antigen gp160, Compared with the current anti-gp41 detection method, the occurrence of missed detection can be reduced; the high-sensitivity urine test of the test strip for detecting HIV antibody in urine of the present invention ensures the authenticity and reliability of the data, and can be used for large-scale primary screening of AIDS.