In recent years, the fluorescence excitation-emission matrix (EEM) technique has become common for chemical analysis, but fluorescent organic matters with similar structures may exhibit extremely similar EEMs, which may mislead the analysis results. Thus, precisely distinguishing the organic matters with similar EEMs is an important problem to solve. Fluorescence quantum yield (FQY) and fluorescence lifetime (FL) are two important optical parameters for EEM, which are more sensitive to the difference in molecular structure. This study investigated the EEM, FQY, and FL of indole, 3-methylindole, and L-tryptophan. The result showed that their EEMs all displayed one emission (Em) maxima corresponding to two excitation (Ex) maxima, and the position is very close. The fluorescence peaks of indole and L-tryptophan were roughly located at Ex/Em=［275, 340～350］ and ［220, 340～350］ nm, the fluorescence peaks of 3-methylindole were roughly located at Ex/Em=［280, 365］ and ［225, 365］ nm. At the same concentration, these three compounds maximum fluorescence intensity (MFI) at Ex = 275~280 nm followed this sequence: indole>3-methylindole>L-tryptophan. The integral sphere technique evaluated the FQYs of indole, 3-methylindole and L-tryptophan as 0.264, 0.347, and 0.145, respectively. The FLs of indole, 3-methylindole and L-tryptophan were determined as 4.149, 7.896 and 2.715 ns, respectively. This study indicated that the FQY and FL can distinguish fluorescent organic matters with similar EEMs. The results are of great value in accurately identifying fluorescent organic matters.