In the current study, we present three designer self-assembling peptides (SAPs) by appending RADA 16-I with epitopes IKVAV, RGD, and YIGSR, which have different net charges and amphiphilic properties at neutral pH. The self-assembly of the designer SAPs is intensively investigated as a function of pH, canion type, and assembly time. The morphologies of the designer SAPs were studied by atomic force microscope. The secondary structure was investigated by circular dichroism. The dynamic viscoelasticity of designer SAP solutions was examined during titration with different alkaline reagents. Our study indicated that both electrostatic and hydrophilic/hydrophobic interactions of the motifs exhibited influences on the self-assembly, consequentially affecting the fiber morphologies and rheological properties. Moreover, NaOH induced a quicker assembly/reassembly of the designer SAPs than Tris because of its strong ionic strength. Therefore, our study gained comprehensive insight into the self-assembling mechanism as references for developing RADA 16-I-based functional SAPs.