Our recent publication illustrates the critical role of phenylalanine-mediated aromatic-aromatic interactions in determining the assembly of peptidic β-sheets. However, the effect of phenylalanine number on regulating the assembly efficacy of peptidic β-sheets remains poorly understood. We herein evaluate the assembly efficacy of β-sheets of a series of oligopeptides which contain 0, 1, 2, or 3 phenylalanine in their molecular backbones. In our assembly system, two phenylalanine (2F) is the minimum number for driving the assembly of β-sheets of oligopeptides. Oligopeptides with three phenylalanine (3F) show significantly increased assembly efficacy of β-sheets compared to that with 2F. These results suggest a positive correlation between the phenylalanine number and assembly efficacy of β-sheets. By improving the assembly efficacy of β-sheets, we further develop a highly sensitive HIV analytical system in which the specific binding of β-sheets with Congo Red induces enhanced fluorescence. For HIV p24 detection, the 3F-based analytical system (0.61 pg/mL) shows a significantly lower limit of detection (LOD) than the 2F-based analytical system (2.44 pg/mL), both of which are more sensitive than commercial ELISA (5 pg/mL) used in the clinic. This work not only illustrates the effect of phenylalanine number on regulating the assembly efficacy of β-sheets but also provides a guideline for the construction of a highly sensitive analytical system of disease diagnosis.