Combining the noncovalent and covalent interactions, a series of peptide amphiphiles were designed de novo and synthesized to architect functional assemblies by means of photochemistry. The strand of peptide sequence was structurally capped with photoactive tyrosine-tyrosine (YY) motifs at both termini, and the spacing was filled by alternating of hydrophilic D (L-aspartate) and hydrophobic X (ε-aminocaproic acid) structure. Upon visible-light irradiation, these de novo designed peptides underwent rapid photocrosslinking within merely 10 min. Interestingly, the modulation of alternating D–X pairs in occupying spacer would adjust molecular amphiphilicity, regulate charge distribution, and control particle size and loading capacity of peptide nanospheres (PNS) in aqueous media. With entirely peptide-based matrix, this PNS system could host cationic indicators of fluorescent rhodamine and magnetic Gd^III for exemplar near infrared (NIR) fluorescence and magnetic resonance (MR) imaging, which paves a pathway to biomaterial and biomedical applications using de novo designed peptides.