A first-principles investigation on the effect of O-doping and W-addition as well as O-W coexistence effect on the Ni/Ni3Al interface in Ni-based alloys is performed. The results reveal that O occupies octahedral interstitial sites while W substitutes for Ni or Al atoms at the Ni/Ni3Al interface. O significantly reduces the interface cohesive strength, while W enhances the cohesion of the Ni/Ni3Al interface. In the cases of O-W co-existence, O and W maintain their individual weakening and strengthening effects, in most cases, the weakening effect of O-doping is more pronounced. When O is located in the coherent (002)γ/γ′ layer, the fracture strength and toughness of the co-doping interface are even worse than when O doped alone. Furthermore, the influence of O-W co-doping on the interface appears insensitive to the atomic distance between O and W. Electronic structure analysis reveals that the embrittling effect of O originates from its local electron aggregation effect, while W results in a strengthening effect at close range and a slight weakening effect at longer distances in the interfacial region. The findings provide insights into the complex effects of multiple elements interactions and suggest a potential strategy for the design of Ni-based alloys.