The invention relates to a pH-responsive type magnetic composite microsphere with a Janus structure, and a preparation method thereof, and belongs to the fields of composite materials and nanotechnologies. The pH-responsive type magnetic composite microsphere is of a core-shell structure, an inner core is made of polystyrene wrapped with Fe3O4, and a shell layer is made of poly(tert-butylmethacrylate). The preparation method comprises the following steps: preparing magnetic Fe3O4 nano-particles, then adding oleic acid, washing to neutral to obtain oleic modified Fe3O4 nano-particles, then dissolving in normal octane, adding a styrene monomer and a tert-butyl methacrylate monomer after ultrasound to obtain a magnetofluid, and adding in a bottle equipped with a reflux condensing tube and a thermometer, adding deionized water, lauryl sodium sulfate and NaHCO3 in sequence, heating in a water bath, then introducing N2, adding ammonium persulfate, preparing a magnetic composite microsphere solution with the Janus structure after emulsion polymerization, then centrifuging, separating a lower sediment, washing to neutral, putting in the container after drying, and adding deionized water and trifluoroacetic acid with different pH values to obtain a product.