Separation effects of sodium polyacrylate (PAAS) and gum Arabic (GA) on flotation of chalcopyrite and magnesium silicate minerals using potassium butyl xanthate (PBX) as collector were investigated by micro-flotation experiments, zeta potential, Infrared spectral (IR), SEM-EDS, XPS analysis and copper sulphide ore beneficiation test. The micro-flotation experiments and zeta potential measurements showed that combined depressant consisting of PAAS and GA could efficiently reduce the recoveries of mixed minerals of serpentine and talc more than 25%, while that of chalcopyrite remained above 70% at pH 9.2. Infrared spectral (IR), SEM-EDS and XPS analysis showed that PAAS chemically reacted with Mg on the surface of serpentine, while GA adsorbed on talc surface mainly via physical interaction and hydrogen bond may also play a role. Surface synergism between PAAS and GA was investigated by turbidity test and its depression mechanism was proposed. The technology feasibility of using PAAS and GA to improve the copper sulphide ore flotation performance was verified through artificial mixed ore flotation and laboratory closed-flotation operation.