Self-assembled nanomaterials show potential high efficiency as theranostics for high-performance bioimaging and disease treatment. However, the superstructures of pre-assembled nanomaterials may change in the complicated physiological conditions, resulting in compromised properties and/or biofunctions. Taking advantage of chemical self-assembly and biomedicine, a new strategy of "in vivo self-assembly" is proposed to in situ construct functional nanomaterials in living subjects to explore new biological effects. Herein, recent advances on peptide-based nanomaterials constructed by the in vivo self-assembly strategy are summarized. Modular peptide building blocks with various functions, such as targeting, self-assembly, tailoring, and biofunctional motifs, are employed for the construction of nanomaterials. Then, self-assembly of these building blocks in living systems to construct various morphologies of nanostructures and corresponding unique biological effects, such as assembly/aggregation-induced retention (AIR), are introduced, followed by their applications in high-performance drug delivery and bioimaging. Finally, an outlook and perspective toward future developments of in vivo self-assembled peptide-based nanomaterials for translational medicine are concluded.