The digital breast tomosynthesis (DBT) has been widely used as a diagnosis imaging modality of breast cancer because of potential for structure noise reduction, better detectability, and less breast compression. Since 3D modulation transfer function (MTF) is one of the quantitative metrics to assess the spatial resolution of medical imaging systems, it is very important to measure 3D MTF of the DBT system to evaluate the resolution performance. In order to do that, Samei et al. used sphere phantoms and applied Thornton’s method to the DBT system. However, due to the limitation of Thornton’s method, the low frequency drop, caused by the limited data acquisition angle and reconstruction filters, was not measured correctly. To overcome this limitation, we propose a Richardson-Lucy (RL) deconvolution based estimation method to measure the directional MTF. We reconstructed point and sphere objects using FDK algorithm within a 40⁰ data acquisition angle. The ideal 3D MTF is obtained by taking Fourier transform of the reconstructed point object, and three directions (i.e., fx-direction, fy-direction, and fxy-direction) of the ideal 3D MTF are used as a reference. To estimate the directional MTF, the plane integrals of the reconstructed and ideal sphere object were calculated and used to estimate the directional PSF using RL deconvolution technique. Finally, the directional MTF was calculated by taking Fourier transform of the estimated PSF. Compared to the previous method, the proposed method showed a good agreement with the ideal directional MTF, especially at low frequency regions.