Revealing the effect of solid solution strengthening in nanocrystalline alloys is challenging because observed mechanical properties are often influenced by several intertwined underlying microstructural factors. This paper designed and prepared a series of multilayered films consisting of 28-nm-thick crystalline layers with low Zr content and 2-nm-thick amorphous layers with high Zr content and through varying Zr content of columnar grains in crystalline layers, achieved the isolation of intragranular solute from other microstructure factors. The results of nanoindentation showed that the effect of solid solution strengthening still existed in Al-Zr alloy when the grain size decreased to 28nm. The strengthening effect was consistent with the Fleischer model for coarse-grained materials.