Tumor has greatly threatened to human life. Now the main means of tumor treatment are surgery and(/or) chemoradiotherapy. However, the chemoradiotherapy is usually not suitable to sustained treatment of tumors due to its poor cell selectivity, side effects and inducing tumor cells resistance. Therefore, it is urgent to develop new drugs with low toxicity and high activity at the tumor sites. In situ self-assembly peptides which can target the tumors and self-assemble into specific nanostructures induced by the specific and highly expressed substances at the tumor tissues are expected to be a new kind of anti-tumor drugs. In this study, we designed a peptide denoted as Fmoc-FFRIKFERQ-OH based on the concept of in situ self-assembly of peptides. This peptide can target lysosomes and self-assemble in situ after being degraded by cathepsin L in lysosomes. The self-assembly properties of Fmoc-FFRIKFERQ-OH and its enzymolysis products degraded by cathepsin L were oberved by AFM and MALDI-TOF-MS. The anti-tumor activity of this peptide was measured by using the MTT and flow cytometry assay. The results demonstrated that cathepsin L could precisely hydrolyze the Fmoc-FFRIKFERQ-OH molecule under acidic conditions in vitro, and its enzymolysis product Fmoc-FFR-OH self-assembled into long nanofibers, which had high antitumor activity against both A375 and SH-SY5Y cells. The relative survival rate was only 36.08% and 25.56% for A375 and SH-SY5Y, respectively. Comparatively, Fmoc-FFRIKFERQ-OH had lower toxic and side effects on normal cells L929. The self-assembled long fibers formed by Fmoc-FFR-OH can disrupt the membrane of lysosome, which can induce cell apoptosis and(/or) death. Thus, the designed Fmoc-FFRIKFERQ-OH is expected to be a new anti-tumor drug due to its high antitumor activity and low toxicity against to normal cells.