The present disclosure discloses a method for isolating and purifying Lactobacillus rhamnosus ZFM216 bacteriocin, the method including the following steps: preparing L. rhamnosus ZFM216 fermentation supernatant; treating the fermentation supernatant with macroporous adsorption resin method to obtain a crude protein extract; isolating the crude protein extract by gel chromatography to obtain a preliminary purified protein solution; and then performing purification by reversed-phase high performance liquid chromatography (HPLC) to obtain an L. rhamnosus ZFM216 bacteriocin solution. The present disclosure further provides use of the L. rhamnosus bacteriocin in anti-Staphylococcus aureus.