The aim of this study is to develop a unified optimization method to estimate the complete parameters about geometric calibration of system misalignment for both cone-beam CT and pinhole SPECT, and parameters about coordinate transformation of image registration for these detached systems. The uniform projection equations are derived for both cone-beam and pinhole imaging geometry. Complete geometric parameters are estimated by point object phantom with a priori relative position information. The cost function is structured as the least-squares about residual error. The implementation of nonlinear estimation utilizes the Powell method so as to constrain the optimization problem of this study to converge to the unique solution. The registration procedure is also implemented by the nonlinear optimization method similar to the geometric calibration, and six parameters are sufficient to register the detached systems for multi-modality image fusion. We designed a general-purpose point object/sources phantom for both geometric calibration and image registration procedures. To validate the method presented, we performed small animal experiment studies based on our Micro-SPECT and Micro-CT systems. The results from the imaging experiments show that the unified estimation method is considerable effective and robust for detached multi-modality Micro-SPECT/CT systems.