The layered structure of a perovskite tricolor superlattice {SrTiO3(STO)∕LaAlO3(LAO)∕La0.6Sr0.4MnO3(LSMO)}n on an STO substrate is investigated using transmission electron microscopy (TEM) and electron energy-loss spectroscopy (EELS). Conventional TEM imaging is not effective for thickness evaluation, in which both LAO and LSMO layers show the same image contrast because of the similarity in their mean atomic numbers. We observe 001 lattice fringes, which are chemically sensitive in the case of the perovskite structure ABO3, since the 001 structure factor is proportional to the difference between A- and B-site atomic scattering factors. The thickness of each layer is evaluated with an accuracy of one unit cell. We also apply spatially resolved EELS to analyze the depth profile of Ti, Mn, and La contents.