Pathological stimuli-responsive self-assembly of peptide nanofibers enables selective accumulation of imaging agent cargos in the stimuli-rich regions of interest. It provides enhanced imaging signals, biocompatibility, and tumor/disease accessibility and retention, thereby promoting smart, precise, and sensitive tumor/disease imaging both in vitro and in vivo. Considering the remarkable significance and recent encouraging breakthroughs of self-assembled peptide nanofibers in tumor/disease diagnosis, this reivew is herein proposed. We emphasize the recent advances particularly in the past three years, and provide an outlook in this field.