The invention discloses a dot fluorescence immunoassay method for quantitatively detecting a plant virus. The dot fluorescence immunoassay method comprises the following steps: grinding a sample to be tested, preparing a sampling solution of the detected sample, preparing a positive control sampling solution, a negative control sampling solution and a blank control sampling solution, measuring a virus particle average number d of the positive control sampling solution; respectively dotting 2.0-2.5 mu l of the sampling solution of the detected sample, the positive control sampling solution, the negative control sampling solution and the blank control sampling solution on a nitrocellulose membrane, closing for 0.8-1.2h after the nitrocellulose membrane is dried, sequentially adding a diluted primary antibody and a diluted secondary antibody, respectively reacting for 0.8-1.2h, washing the membrane for 3-4 times after each reaction is performed, wherein the washing of each time is performed for 5-6 min; and after slightly drying the membrane, detecting fluorescence intensity m in a fluorospectro photometer, and calculating a particle average number d of a virus to be tested in the sampling solution of the detected sample according to the following formula. A ratio of a value m of the positive control sampling solution to a value d of the positive control sampling solution is equal to a ratio of a value m of the sampling solution of the detected sample to a value d of the sampling solution of the detected sample. The dot fluorescence immunoassay method is capable of realizing sensitive, accurate, rapid and efficient quantitative analysis of the plant virus, and is high in popularization and application values.