The invention discloses a flotation separation method of high-sulfur and high-arsenic gold ore, and belongs to the technical field of mineral processing. The flotation separation method comprises thesteps of (1) grinding raw ore to the preset fineness; (2) performing shaking table sorting treatment on ground products so as to recycle natural and native gold dissociated from the raw ore; and (3) merging shaking table tailings, then performing flotation operation, adding water glass and macromolecular agents containing -OH and -O- groups as a combined inhibitor, adding copper sulfate as an activating agent, fully mixing, then adding a collecting agent and a foaming agent, and performing flotation separation so as to obtain gold concentrates. According to the flotation separation method of the high-sulfur and high-arsenic gold ore, gold is recycled by adopting a reselection-flotation combined technological process, so that the recycling of gold is strengthened; the macromolecular agentscontaining the -OH and -O- groups are added, so that the inhibiting effects on chlorite, steatite and other laminar silicate mineral gangue minerals are enhanced, efficient separation of gold carryingminerals and gangue minerals is realized, and the grade of the gold concentrates is guaranteed. The process has the advantages of operation simplicity and high adaptability to ore and can satisfy the requirements of industrial production.