Featured Application: The NIRF-TGI-CT presented in this study is expected to be applied to the acquisition of information, that is, temperature, on the deep interior of samples, especially biological tissues possessing refractive index interfaces in inside that cause light scattering. We report a computed tomography (CT) technique for mapping near-infrared fluorescence (NIRF) lifetime as a multiplex three-dimensional (3D) imaging method, using a conventional NIR camera. This method is achieved by using a time-gated system composed of a pulsed laser and an NIR camera synchronized with a rotatable sample stage for NIRF-CT imaging. The fluorescence lifetimes in microsecond-order of lanthanides were mapped on reconstructed cross-sectional and 3D images, via back-projection of two-dimensional projected images acquired from multiple angles at each time point showing fluorescence decay. A method to select slopes (the observed decay rates in time-gated imaging) used for the lifetime calculation, termed as the slope comparison method, was developed for the accurate calculation of each pixel, resulting in reduction of image acquisition time. Time-gated NIRF-CT provides a novel choice for multiplex 3D observation of deep tissues in biology.