The invention discloses an artificial insemination instrument for queen bees with a precision three-dimensional guide rail and a using method thereof, and belongs to the professional technical field of honeybee breeding. The artificial insemination instrument comprises a ventral hook control rod, a back hook control rod, a microsyringe, a polyethylene syringe needle, upper and lower guide rails, front and rear guide rails, left and right guide rails, a spherical steering valve I, a magnetic type anesthetic room, a stand column I, a base, an anti-skid pad, a stand column II and a spherical steering valve II. The stand column I and the stand column II are arranged on the left side and the right side of the upper portion of the base respectively. The ventral hook control rod and the stand column I are connected through the spherical steering valve I arranged on the stand column I, the back hook control rod and the stand column II are connected through the spherical steering valve II arranged on the stand column II, the upper and lower guide rails, the front and rear guide rails and the left and right guide rails are combined into the three-dimensional guide rail, the three-dimensional guide rail is arranged on the stand column II, and the microsyringe is fixedly arranged on the upper and lower guide rails. According to the artificial insemination instrument for queen bees, on the premise of not damaging queen bees, artificial insemination of the queen bees can be precisely completed, the insemination precision is improved, operation is convenient, and the service life is prolonged.