An assembly-induced retention effect for enhanced tumor photoacoustic (PA) imaging and therapeutics is described. A responsive small-molecule precursor is prepared that simultaneously self-assembles into nanofibers in tumor sites that exhibit an assembly-induced retention effect, which results in an improved PA imaging signal and enhanced therapeutic efficacy. This successful proof-of-concept study paves the way to develop novel supramolecular biomaterials for cancer diagnostics and therapeutics. As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.