We synthesized multiple-cation Rb(MAFA)PbI3 perovskite single crystals for the first time. The effect of Rb+ substitution was systemically investigated, and the addition of 1.5 M 5% RbI was the optimum condition to obtain high-quality Rb(MAFA)PbI3 single crystals. Lattice shrinkage occurred in the Rb(MAFA)PbI3 single crystal because of the small ionic radius of Rb+, resulting in blue-shifted absorption and photoluminescence (PL) peaks. The 1.5 M 5% RbI-added (MAFA)PbI3 single crystal showed the longest carrier lifetime of 18.35 ns, exhibiting the highest photoresponse than other crystals. We believe that this work will provide a basic insight into the mixed-cation perovskite single crystals for the future optoelectronic applications.