An optical tweezers controlling device including a light source, an objective lens and a focus adjusting unit is provided. The focus adjusting unit disposed between the light source and the objective lens includes a mirror set and a zoom lens set. The mirror set has at least a mirror. The mirror is rotatable such that after a light of the light source is projected to the mirror, the reflective direction of the light reflected from the mirror is changeable. The zoom lens set has at least a zoom lens disposed in accordance with the mirror. By rotating the mirror or changing the focal length of the zoom lens, the focusing location of the light changes on the focal plane of the objective lens or in the front or the rear of the focal plane.