This study evaluated 8 unstained human coronary tissues ex vivo using spectrum- and time-resolved multiphoton microscopy. First, according to the spectra, the elastin fibers and collagen fibers in the coronary arterial intima can be separated clearly. Second, the ratios of the two types of fiber signals were calculated to assess changes in the relative content of the collagen and elastin fibers in the coronary arterial wall caused by an atherosclerotic lesion. Third, we assessed biochemical variations of the elastin fibers in the coronary atherosclerotic tissues by measuring fluorescence lifetime and found that the coronary atherosclerotic plaque has a lower mean fluorescence lifetime than a normal tissue. This study demonstrates that spectrum- and time-resolved multiphoton microscopy can effectively identify coronary atherosclerotic plaques, thus indicating its potential as a novel research tool for studying coronary arteriosclerotic lesions in the future.