A microfluidic control apparatus and operating method thereof. The microfluidic control apparatus includes a photoconductive material layer and a flow passage. When a light with a specific optical pattern is emitted toward the photoconductive material layer, at least three virtual electrodes are formed on the photoconductive material layer according to the specific optical pattern. The at least three virtual electrodes include a first virtual electrode, a second virtual electrode and a third virtual electrode disposed beside the first virtual electrode. There is a specific proportion among a distance between first virtual electrode and third virtual electrode, a width of first virtual electrode, a distance between first virtual electrode and second virtual electrode, and a width of second virtual electrode. When the specific optical pattern changes, the at least three virtual electrodes also change to generate an electro-osmotic force to control the moving state of a microfluid in a flow passage.