Background: The precise assessment of myocardial infarction (MI) is crucial both for therapeutic interventions in old MI and the development of new and effective techniques to repair injured myocardium. A novel method was developed to assess left ventricular (LV) quantitatively infarction through three-dimensional (3D) multimodality fusion based on computed tomography angiography (CTA) and technetium-99m methoxyisobutylisonitrile (99mTc-MIBI) single-photon emission computed tomography (SPECT) images. This study sought to develop a 3D quantitative method for MI for pre-clinical study and clinical application. Methods: Three months after the MI models were established in 20 minipigs, CTA and SPECT images were acquired separately, which were then aligned automatically with the constraints of the shape and the whole heart and LV myocardium position. Infarct ratios were quantified based on the 3D fusion images. The quantitative assessment was then experimentally validated via an ex vivo histology analysis using triphenyl-tetrazolium-chloride staining and subsequently applied to post-MI patients (n=8). Results: The location of an infarct identified by the SPECT was consistent with that identified by an ex vivo heart in a 3D space. Infarct size determined by CTA-SPECT was correlated with infarct size assessed by triphenyl-tetrazolium-chloride pathology (27.6% [interquartile range (IQR) 17.1-34.7%] vs. 24.1% (IQR 14.7-32.5%), r2=0.99, P<0.01). In clinical cases, the CTA-SPECT 3D fusion quantitative results were significantly correlated with the quantitative perfusion SPECT results (r=0.976, P<0.01). Conclusions: The proposed 3D fusion quantitative assessment method provides reliable and intuitive evaluations of infarction. This novel quantification technique enables whole heart quantification for the pre-operation evaluation and post-diagnosis management of old MI patients. It could also be applied to the design of 3D-printed cardiac patches.