The present study aimed to develop a monoclonal antibody (mAb)-based double functional probe for PET and near-infrared fluorescence (NIRF) targeting CD20 and to cross validate the targeting efficacy of this dual functional probe. NuB2, anti CD20 mAb was conjugated with Alexa Fluor 750 and 64Cu through DOTA chelator. PET and NIRF imaging was carried out at 30 min and 24 h post-injection with 64Cu-DOTA-NuB2-Alexa Fluor 750 in CD20 positive Raji lymphoma-bearing mice. Fluorescence intensity and radio-activity were studied by ex vivo biodistribution study at 30 min, 24 and 48 h after injection. Raji tumor showed significantly higher uptake of DOTA-NuB2-Alexa Fluor 750 than that of DOTA-Alexa Fluor 750 (p<0.05). Significant correlation was obtained between the organ-to-muscle ratios measured by the radioactivity and fluorescence intensity (p<2.2e-16, r=0.94). Our findings demonstrate the effectiveness and feasibility of preparing an effective mAb-based dual functional imaging agent for PET and NIRF targeting the CD20 expression in lymphoma.