An alignment method for assembling substrates in different spaces without fiducial mark and its system are provided, and the alignment method has steps of: pre-defining partially standard character regions of two substrates; capturing at least two partially actual images of two substrates in different waiting spaces, respectively; comparing to obtain at least two partially actual character regions of the two substrates, respectively; building actual coordinate systems of the two substrates, respectively; comparing the actual coordinate systems of the two substrates with each other to obtain a set of offset values; moving the two substrates from the different waiting spaces to an alignment-and-installation space based on the set of offset values and a predetermined movement value, respectively; and stacking the two substrates with each other to finish the alignment and installation in the alignment-and-installation space.