The invention relates to a fluorescence imaging detection device, and particularly provides a directional emission fluorescence imaging detection device, which is provided with an optical laser beam-expanding system, a sample system and a detecting system, wherein the optical laser beam-expanding system is provided with a laser light source, a microobjective, a needle hole, a plano-convex lens, an attenuator and a rectangular diaphragm, the excitation light emitted by the laser light source sequentially passes by the microobjective, the needle hole and the plano-convex lens for beam expanding and collimation to form parallel light beams, and the parallel light beams sequentially pass by the attenuator and the diaphragm to obtain light spots; the sample system is provided with a rotating stage, a sample rack, a prism and an optical quartz substrate, the prism is placed on the sample rack and fixed at the center of the rotating stage, a nanoscale metallic film is arranged on the surface of the quartz substrate, a fluorescence sensing film is arranged on the surface of the metallic film, and solution is coated between the quartz substrate and the prism; and the detecting system is provided with a CCD rotating arm, a CCD receiver and an optical filter, the CCD rotating arm is fixed on the stage board of the rotating stage, the CCD rotating arm is connected onto the stage board of the rotating stage, and the CCD receiver used for collecting images and the optical filter are arranged on the CCD rotating arm.