The invention relates to a nano probe used for nucleolin cross-linking to induce the tumor cell apoptosis, and a preparation method and application of the nano probe, and belongs to the technical field of biology. The nano probe uses ethylene glycol chitosan as a polymer backbone, the polymer backbone is grafted with cholesterol and an A chain, the 5'end of the A chain is modified with a carboxylgroup, the 3'end of the A chain is modified with a dye Cy3, the A chain contains a nucleic acid adapter AS1411 gene sequence, and the A chain partially hybridizes with a B chain modified with BHQ2 atthe 5'end. The nano probe can selectively kill tumor cells with the overexpression of nucleolin, but has relatively little effects on normal cells, is expected to become a method for the treatment ofmalignant tumors, and provides a new alternative receptor for the idea of receptor cross-linking to induce apoptosis. The nano probe is simple in synthesis, separation and purification, and is suitable for expanded production.