Two-photon fluorescence imaging technology has several outstanding characteristics, including lower photon-bleaching and photo-damaging, high contrast and longer penetrating distance. An acute myocardial ischemia model of rat is established by the ligation of the left anterior descending branch. Then fluorescence images of the front wall of left ventricle at different ligation time are attained by two-photon fluorescence imaging. The morphologic change of myocardial ischemia tissue was obtained and a primary evaluation of the damage degree of myocardial tissue is obtained by fast Fourier transform (FFT) with an orientation index. The result indicates that the two-photon fluorescence imaging technology combined with FFT has the potential to be used for the estimation of myocardial ischemia tissue range and assess of the damage degree of myocardial tissue rapidly and accurately in the diagnosis and therapy in real time.