Based on the self-assembly process occurring in the human body all the time, self-assembled nanomaterials were designed by the researchers. The self-assembled nanomaterials have controllability, biocompatibility and functional advantages in vivo. The self-assembled nanomaterials constructed in situ under a physiological environment display various biological characteristics which can be used for imaging, therapy, and broad clinical applications. In situ self-assembled nanomaterials can boost drug function, reduce toxic and side effects, prolong imaging time and enlarge signal-to-noise ratio. By using pathological conditions to trigger specific responses in vivo, well-ordered nanoaggregates can be spontaneously formed by multiple weak bonding interactions. The assembly shows higher accumulation and longer retention in situ. Endogenous triggers for in situ assembly, such as enzymes, pH, reactive oxygen species and ligand receptor interaction, can be used to transform the materials into a variety of controllable nanostructures including nanoparticles, nanofibers and gels through bioactivated in vivo assembly (BIVA) strategies. BIVA strategies can be applied for treatment, imaging or participate in the physiological activities of cells at the lesion site. This review summarized and prospected the design of self-assembled peptide materials based on BIVA technology and their biomedical applications. The nanostructures of the self-assembly enable some beneficial biological effects, such as assembly induced retention (AIR) effect, enhanced targeting effect, multivalent bond effect, and membrane disturbance. Thus, the BIVA nanotechnology is promising for efficient drug delivery, enhancement of targeting and treatment, as well as optimization of the biological distribution of drugs.