Self-assembled nanofibers hold tremendous promise for cancer theranostics owing to their in situ assembly, spatiotemporal responsiveness, and diverse bioactivity. Herein, this review summarizes the recent advances of self-assembled peptide nanofibers and their applications in biological systems, focusing on the dynamic process of capturing cancer cells from the outside-in. (1) In situ self-assembly in response to pathological or physiological changes. (2) Diverse functions at different locations of tumors, such as forming thrombus in tumor vasculature, constructing a barrier on the cancer cell membrane, and disrupting the cancer organelles. Of note, with the assembly/aggregation induced residence (AIR) effect, the nanofibers could form a drug depot in situ for sustained release of chemotherapeutic drugs to increase their local concentration and prolong the residence time. Finally, perspectives toward future directions and challenges are presented to further understand and expand this exciting field.