The invention discloses a near-infrared nanoprobe, a preparation method and a holographic molecular imaging navigation system. The preparation method of the near-infrared nanoprobe comprises the steps that a mixed solution of near-infrared fluorescent dyes is obtained; iodinated oil injection is added into the mixed solution; an organic solvent in the mixed solution where the iodinated oil injection is added is volatilized, and an iodinated oil mixed solution is obtained; a nano surface active agent is dissolved into absolute ethyl alcohol, and a nano surface active agent solution is obtained; the nano surface active agent solution and the iodinated oil mixed solution are added into a polyethylene glycol solution, and a nano preparation is formed; and the nano preparation is filtered, and the near-infrared nanoprobe is obtained. The physical and chemical properties of the near-infrared nanoprobe are more stable, and an excellent fluorescence enhancing effect and biological stability are achieved; and meanwhile the assorted holographic molecular imaging navigation system is combined, a cancer area can be clearly recognized, and the liver cancer boundary is determined, so that precise excision of a cancer lesion is achieved.