A microfluidic device with microstructure includes a channel for accommodating an electrolytic solution therein and at least one microstructure formed in the channel. When an alternating-current signal is input to the microfluidic device so that a surface of the microstructure is polarized by a generated electric field, ions having polarity reverse to that of an electrolytic solution will migrate to the surface of the microstructure to form a field-induced electrical double layer to result in electro-osmotic flows at the corners at two sides of the microstructure, which causes formation of relatively fierce circular vortices in the solution. A sensing system and a sensing method using the microfluidic device with microstructure are also disclosed.