The invention discloses a preparation method of an immune-magnetic bead. The preparation method comprises the following steps: firstly, dispersing freeze-dried silicon-based magnetic beads in a polar organic solvent, treating with ultrasonic wave, subsequently adding a dispersant dissolved in the polar organic solvent, stirring, dripping organic small molecules which are dissolved in the polar organic solvent and contain double-end carboxyl, and then reacting for 6-18 hours after the dripping is ended; magnetically separating out carboxyl magnetic beads after the reaction ends, sequentially and repeatedly washing precipitates for a plurality of times by use of absolute ethyl alcohol and deionized water until the pH value of a washing liquid is 6-7, performing ultrasonic treatment for 15-30 minutes, and dispersing into 0.02% of NaN3 water to obtain the carboxyl magnetic beads; washing the carboxyl magnetic beads by use of an MES (Methyl Ester Sulfonate) buffer solution at least once, re-suspending the carboxyl magnetic beads in the MES buffer solution, activating the magnetic beads by using an activating agent carbodiimide, and then coupling an antibody in the environment of the MES buffer solution. The preparation method disclosed by the invention is simple in technological process, simple and convenient to operate and suitable for batch production.