In vivo electroporation (EP) is an efficient method for effective gene transfer and is highly expected for application in anticancer gene therapy. Non-invasive monitoring of gene transfer/expression is critical for optimal gene therapy. Here we report in vivo optical and high-field magnetic resonance imaging (MRI) of EP-mediated transgene expression in a tumor model. Initially, we observed spatio-temporal change in in vivo EP-mediated transgene expression by optical imaging using red fluorescence protein (RFP) as a reporter gene. Next, we constructed a dual-reporter plasmid carrying a gene-encoding MRI reporter ferritin heavy chain and RFP gene to visualize the intratumoral transgene expression by dual modality. Cells transfected with this plasmid showed lower signal intensity on in vitro T(2)-weighted cellular MRI and quantitatively increased the transverse relaxation rate (1/T(2)) compared with control cells. After conducting in vivo EP in an experimental tumor, the plasmid-injected region showed both fluorescent emissions in optical imaging and detectably lowered signal on T(2)-weighted MRI. The correlative immunohistological findings confirmed that both the reporter transgenes were co-expressed in this region. Thus, our strategy provides a platform for evaluating EP-mediated cancer gene therapy easily and safely without administering contrast agent or substrate.