A semiconductor structure includes: a plurality of calibration reference features disposed on a substrate and spaced apart from each other in a first direction; and a plurality of columns of first active features and a plurality of columns of second active features respectively disposed on opposite sides of the calibration reference features, wherein each of the columns of first active features is spaced apart from each other in a second direction, each of the columns of second active features is spaced apart from each other in the second direction, and the calibration reference features, the first active features, and the second active features are disposed on the same layer and are a portion of the substrate.