Retinal surgery is a complex activity that can be challenging for a surgeon to perform effectively and safely. Image guided robot-assisted surgery is one of the promising solutions that bring significant surgical enhancement in treatment outcome and reduce the physical limitations of human surgeons. In this paper, we demonstrate a novel method for 3D guidance of the instrument based on the projection of spotlight in the single microscope images. The spotlight projection mechanism is firstly analyzed and modeled with a projection on both a plane and a sphere surface. To test the feasibility of the proposed method, a light fiber is integrated into the instrument which is driven by the Steady-Hand Eye Robot (SHER). The spot of light is segmented and tracked on a phantom retina using the proposed algorithm. The static calibration and dynamic test results both show that the proposed method can easily archive 0.5 mm of tip-to-surface distance which is within the clinically acceptable accuracy for intraocular visual guidance.