The invention discloses a preparation method of mulberry silk capable of fluorescing under irradiation of near-infrared light, including the steps of (1), preparing upconversion nanoparticles, performing surface modification with concanavalin to obtain modified upconversion nanoparticles; (2), uniformly dispersing the upconversion nanoparticles in water to prepare aqueous solution of upconversionnanoparticles; (3), picking mature mulberry leaves, soaking the mulberry leaves in the aqueous solution of upconversion nanoparticles, draining water and then drying in air naturally; (4), feeding silkworms the processed mulberry leaves until the silkworms spin and make cocoons when the silkworms grow to the designed stage; (5), collecting silk so as to obtain the mulberry silk capable of fluorescing under irradiation of near-infrared light. According to the preparation method, the upconversion nanoparticles applied to preparation is capable of fluorescing under irradiation of near-infrared light having higher penetrating power, and accordingly has better application in imaging of deep tissue. Besides, the upconversion nanoparticles have much stable property, higher bio-security, higher signal to noise ratio and wider application range.