Fluorescence lifetime imaging can provide valuable diagnostic information relating to the functional status of diseases. In this study, a near-infrared (NIR) dye-labeled hexapeptide (abbreviated Cyp-GRD) was synthesized. In vitro, Cyp-GRD internalized in nonsmall cell lung cancer cells (A549) without observable cytotoxic or proliferative effects to the cells at a concentration up to . Time-domain fluorescence intensity and lifetime imaging of Cyp-GRD injected into A549 tumor-bearing mice revealed that the probe preferentially accumulated in the tumor and the major excretion organs. The fluorescence lifetime of the conjugate at the tumor site was mapped, showing the spatial distribution of the lifetime related to its environment. Additionally, fluorescence intensity image reconstruction obtained by integrating the time-resolved intensities enabled the contrast ratios of tumor-to-kidney or liver in slices at different depths to be displayed. The mean lifetime was for the tumor and for the liver when averaging those pixels exhibiting adequate signal-to-noise ratio, showing the tumor had a higher lifetime average and reflecting the altered physiopathology of the tumor. This study clearly demonstrated the feasibility of whole-body NIR fluorescence lifetime imaging for tumor localization and its spatial functional status in living small animals.