A handheld molecular imaging navigation system comprises a multi-spectral light source module configured to provide light in a plurality of different spectrum bands in a time division control manner according to a control signal sequence to irradiate an inspected object; a time division control module configured to generate the control signal sequence; an optical signal acquisition module configured to acquire a near-infrared fluorescence image and a visible light image of the inspected object in a time division control manner according to the control signal sequence provided by the time division control module; and a processing module configured to perform image processing on the acquired near-infrared fluorescence image and visible light image according to the control signal sequence to fuse the visible light image and the fluorescence image and output the fused image, and output a feedback signal according to the acquired near-infrared fluorescence image and visible light image.