The aim of this ex vivo study was to explore the potential of using the fluorescence lifetime of intracellular reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) as a label-free indicator to characterize the differences between human leukemic myeloid cells and normal mononuclear cells (MNC). The steady-state and time-resolved autofluorescence of two human leukemic myeloid cell lines (K562, HL60) and MNC were measured by a spectrofluorimeter. According to excitation–emission matrix (EEM) analysis, the optimal emission of NAD(P)H in these cells suspensions occurred at 445 nm. Furthermore, the fluorescence lifetimes of NAD(P)H in leukemic myeloid cells and MNC were determined by fitting the time-resolved autofluorescence data. The mean fluorescence lifetimes of NAD(P)H in K562, HL60, and MNC cells were 5.57± 1.19, 4.45 ± 0.71, and 7.31 ± 0.60 ns, respectively. There was a significant difference in the mean lifetime of NAD(P)H between leukemic myeloid cells and MNC (p < 0:05). The difference was essentially caused by the change in relative concentration of free and protein-bound NAD(P)H. This study suggests that the mean fluorescence lifetime of NAD(P)H might be a potential labelfree indicator for differentiating leukemic myeloid cells from MNC.