Medical images from different modalities, such as X-ray computerized tomography (CT), magnetic resonance imaging (MRI), single photon emission computed tomography (SPECT) and positron emission tomography (PET) are simultaneously used for clinical diagnosis and research. The intention to integrate medical images includes (1) complementing functional information from functional images with anatomical information from CT and MRI, (2) finding changes over time or under different conditions, or (3) integrating information taken from different imaging modalities. In this paper, we present a three-dimensional (3D) image deformation technique to integrate multi-modality medical images. The 3D optical flow (OF) estimation and thin-plate spline (TPS) transformation are applied on landmarks tracing and geometric deformation, respectively, for inter- and intra-subject medical image integration. The proposed technique is separated into two parts—coarse and fine registration. In the coarse registration, the 3D OF method iteratively estimates moving vectors between corresponding pixels in two image datasets. The desired landmarks are obtained according to the estimated vectors, which relate to the corresponding image dataset, estimated by 3D OF method. As the pairs of landmarks are obtained, these reference landmarks are input into the 3D TPS transformation matrix to generate desired deformation results of coarse image registration. The coarse registration result is applied for fine adjustment by 3D OF estimation by iterative multi-resolution procedures at different levels. To validate the integration performance, the normal brain templates, including MRI and PET volume images, are applied as standard datasets in this study, and the clinical physician selects the standard landmarks from the standard images used for 3D TPS deformation. The results show that RMSE between standard templates and deformation results of MRI and PET studies are less than 3% on average, respectively. In conclusion, the automatic landmarks tracing technique (using 3D OF) and image deformation (using TPS transformation) can minimize human interaction, and integrate medical images effectively.