In this work, the hardness of Ag/Nb/Cu/Nb multilayers with Nb amorphous interlayer were investigated as the individual layer thickness of Ag or Cu (hAg or hCu) varies from 5 to 120 nm. The hardness values of Ag/Nb/Cu/Nb multilayers improve a lot compared with those of the Ag/Cu multilayers and the length scale effect of strengthening becomes broadening in the range of 5–60 nm. At hAg = 60 nm, the hardness remains high and reaches 95% of the maximum hardness of hAg = 5 nm. The enhancement can be contributed to the modulus mismatch and the crystal/amorphous interfaces (CAIs) due to the addition of Nb amorphous interlayer. These CAIs can both increase resistance of dislocations transmission and inhibit the deformation of the amorphous layer.