The invention discloses a chitosan and chitosan quaternary ammonium salt composite magnetic microsphere and a preparation method thereof. The method comprises the steps of preparing a water phase, stirring and dissolving chitosan, chitosan quaternary ammonium salt and a pore-foaming agent PEG or PVP according to a ratio in an acetic acid aqueous solution, and adding magnetic particles for stirring and ultrasonic treatment; preparing an oil phase, and stirring liquid paraffin and Span80 to obtain the oil phase; gradually adding the water phase into the oil phase drop by drop while stirring is carried out to obtain a liquid mixture; adding a sodium polyphosphate aqueous solution into the liquid mixture drop by drop, carrying out stirring, and adding a glutaraldehyde aqueous solution for reaction; carrying out centrifugation and washing to obtain the chitosan and chitosan quaternary ammonium salt composite magnetic microsphere. The composite magnetic microsphere disclosed by the invention is higher in adsorption to electronegative materials such as humic acid, anionic dyes, arsenate ions, phosphate anions and nitrate ions, is higher in pH adaptability, magnetic separation property and regeneration performance, and is high in exchange capacity, high in adsorption speed and easy to separate.