The strengthen separation mechanism of pyrrhotite and chlorite by ultrasonic pre-treatment was revealed through ICP measurements, XPS measurements, SEM analyses, Zeta potential measurements, turbidity analyses, microscopic observation, adsorption capacity measurements, FTIR analyses and contact angle measurements. The results showed that ultrasonic pre-treatment can promote the dissolution of iron oxide particles on pyrrhotite surface and the desorption of iron ions adsorbed on chlorite surface in mixed minerals, which caused Zeta potential and point of zero charge(PZC) negatively shift on pyrrhotite/chlorite surface and convert mutual attraction energy to mutual repulsive energy, thus weaken the hetero-aggregation and enhance the separation between pyrrhotite and chlorite. Ultrasonic pre-treatment can optimize the adsorption capacity of butyl xanthate(BX) and the surface wettability on pyrrhotite surface, however, it has little effect on BX adsorption and wettability on chlorite surface, hence ultrasonic pre-treatment also can strengthen the separation of pyrrhotite from chlorite. Then the flotation experiment of artificial mixed pyrrhotite and chlorite was carried out, the results show that the recovery of pyrrhotite increases from 77.50 % to 81.72 % and the content of MgO decreases from 6.57 % to 5.74 % by the reaction of ultrasonic pre-treatment. Which further proof that ultrasonic pre-treatment can enhance the flotation separation of pyrrhotite from chlorite.