Utilizing molecular dynamics (MD) simulation, the effects of grain size and twin boundary spacings (TBS) on the deformation mechanisms of nano-twinned (NT) Ag-doped Cu alloys are investigated. The doped Ag atoms are segregated to grain boundary affect zone (GBAZ). Both lattice and twining partial dislocations emitted from the amorphous inter-granular layer (GBAZ-segregated domains) are frequently observed, which switch on the strength enhancement and strength softening regime respectively. Consequently, it is hard to relate flow stress with dislocation density. Fortunately, the average flow stress of NT Ag-doped Cu alloy seems to be sensitive to GBAZ phase transition rather than dislocation density.