The invention relates to a preparation method of a hyaluronic acid targeted carbon nano-tube loaded anti-cancer medicine. The preparation method disclosed by the invention comprises the following steps: (1), chemically bonding a multi-walled carbon nano-tube (MWCNT) with PEI (Polyetherimide) in dimethyl sulfoxide through EDC (Ethylene Dichloride), dialyzing, freeze-drying, and synthesizing MWCNT/PEI; (2), dissolving hyaluronic acid in water, and activating by adding EDC; (3), adding FITC (Fluorescein Isothiocyanate) into MWCNT/PEI to stir, adding activated hyaluronic acid (HA), dialyzing, freeze-drying, and synthesizing MWCNT/PEI-FI-HA; and (4), dissolving the MWCNT/PEI-FI-HA in water, adding an aqueous solution of DOX (Doxycycline), adjusting the pH value, sufficiently stirring, dialyzing, and freeze-drying, thus obtaining a medicine loading compound. The preparation method disclosed by the invention is simple in process and easy to operate; the experiment is carried out at normal temperature and normal pressure; after being loaded with the DOX, the hyaluronic acid targeted carbon nano-tube loaded anti-cancer medicine prepared by the preparation method disclosed by the invention has the effects of releasing and actively locating cells in a pH sensitive manner, and therefore, the preparation method disclosed by the invention has a good practical value.