A manufacturing method of microstructure comprises steps of: a motion determination step which determines the motion of a substrate relative to at least a photomask; a microlens determination step which determines the profile of a microlens unit on the substrate; an analysis step which calculates the feature of the photomask according to the motion of the substrate and the profile of the microlens unit by using a numerical analysis method; a production step which produces the photomask according to the feature of the photomask; driving the substrate to do the motion determined in the motion determination step, and meanwhile making a laser light illuminate the substrate through the photomask to manufacture the microlens unit on the substrate by the superposition effect of the laser light; and performing a photolithography process by using the microlens unit to produce a microstructure on a photoresist substrate.