The relative legumain‐targeting efficiency of a Y‐shaped peptide and a linear peptide with the same amino acid sequence was investigated using in vitro experiments and computer simulations. Flow cytometry and fluorescence microscopy of cell types with varying levels of legumain expression (RAW 264.7, 4T1, MCF 10 A, MCF 7 and MDA MB 231) revealed that the linear peptide has a higher legumain binding efficiency in legumain‐active cells compared to the Y‐shaped peptide. Peptide‐protein docking simulations showed that the more stable linear peptide binds at the active site of legumain, whereas the Y‐shaped peptide binds at a different site. The Y‐shaped peptide has its asparaginyl binding site (‐Asn‐) coiled into an α‐helix form, and this reduces access to legumain binding. The linear peptide conjugated to fluorescent carbon dots (CDs) displayed enhanced binding efficiency towards legumain. The peptide‐CDs conjugate nanoparticles are stable under pH, temperature, and medium composition conditions similar to that may be expected in tumor environments, suggesting their high potential as a bioimaging agent.