Abstract: For the detection of TMV in tobacco, antiserum was prepared by injecting purified TMV virion into mouse and the titer of antiserum was determined after adsorption purification, an ELISA analysis system was established basing on the in-house TMV antiserum. The results of PAGE indicated the extracted TMV virion was of high purity and concentration; non-specific antibody could be effectively diminished by adsorption with tobacco total protein; the titer of antiserum was as high as 1:40960; the concentrations of reagents in ELISA detection optimized by orthogonal continuous dilution method were 1:20000 in dilution for secondary antibody, 1:2000 for TMV antiserum and 1:20 for sample supernatant.