The invention discloses a fluorescent staining method for a cotton fiber. The method comprises the specific steps of pretreatment, modification, staining and fixation, wherein in the pretreatment step, a cloth sample is pretreated; in the modification step, water is added and then a modifier is added to the pretreated cloth sample to ensure that original negative charge on the surface of the cotton fiber is changed to positive charge, operation is conducted for 5min, temperature is raised to 70 DEG C at 1.5 DEG C per minute and kept for 30min, the cloth sample is taken out of a cylinder and washed with water once, and the modification is finished; in the staining step, water is added and then fluorescent paint for printing is added according to a color formula to the modified cloth sample, operation is conducted for 5min, temperature is raised to 70 DEG C at 1.5 DEG C per minute and kept for 30min, the cloth sample is taken out of the cylinder and washed with the water once, and the staining is finished; and in the fixation step, water is added and then an adhesive is added to the stained cloth sample, temperature rise and heat preservation are conducted, and the cloth sample is taken out of the cylinder, washed with the water cleanly, softened and dried. According to the fluorescent staining method for the cotton fiber, the fluorescent paint for the printing can be used for staining, and the hand feeling, fastness and cloth cover uniformity are improved.