The invention discloses a bacteriophage able to specifically and efficiently kill multiple resistant staphylococcus aureus, a separation method and application thereof. According to the invention, the bacteriophage able to specifically and efficiently split multiple resistant staphylococcus aureus is screened out from the environment, and the bacteria splitting spectrum is wide. On the basis of a two-layer agar method, a large number of bacteriophages with bacteria splitting activity can be obtained, and the bacteria splitting activity, bacteria splitting spectrum, in vitro bacteria splitting ability and physicochemical properties of the bacteriophages can be determined. The bacteriophage has strong specificity, is difficult to make bacteria generate resistance, also does not produce adverse effect on hosts, and is a feasible method for solving the increasingly serious bacterial resistance problem. The bacteriophage provided by the invention is expected to become a novel antibacterial preparation or environmental disinfectant for preventing and controlling resistant staphylococcus aureus infection.