We report in this study on optimized ratiometric fluorescent probes by peptide self-assembly. The resulting self-assembled nanoprobes show extraordinary stability in aqueous solutions and extremely low background fluorescence in buffer solutions. Our optimized probes with much bigger ratiometric fluorescence ratios also show an enhanced cellular uptake, lower background noise, and much brighter fluorescence signal in the cell experiment. Our study provides a versatile and very useful strategy to design and produce fluorescent probes with better performance.