The oxidative leaching mechanism of sphalerite in ammoniacal solution was investigated by the combination of experimental method and first-principle calculations based on density functional theory. The interaction machanism of NH3, O2, and Na2S2O8 with the ZnS (110) surface was investigated by first-principle calculations. The NH3 molecule preferred to interact with the ZnS (110) surface at TZn with an interaction energy of −366.17 kJ/mol. The active site changed to BZn site on the ZnS (110) surface with the addition of oxidant (O2 or Na2S2O8). The ZnS bond on the ZnS (110) surface was dissociated, where S2− was oxidized into S22−. Zn extraction was enhanced by the addition of the oxidants. In water, S2O82− is more active than the O2 molecule to interact with the ZnS (110) surface with an interactional energy of −588.77 kJ/mol. The effects of the kinds and amount of the oxidant on the Zn extraction were determined under optimum leaching condition ([NH3]: [NH4+] = 5:3, liquid to solid ratio = 25, temperature = 383 K and leaching time = 4 h). Experiment results showed that Zn extraction was significantly enhanced from 4.32% to 70.30% within 4 h at O2 partial pressure of 0.50 MPa or to 28.40% with the addition of persulfate.