We described a novel handheld device (termed FMTPen) for three-dimensional (3D) fluorescence molecular tomography (FMT). The FMTpen is characterized by its bendable structure and miniaturized size (10 mm in diameter) that can be potentially used as an intraoperative tool for the detection of tumor margins and for image-guided surgery. Several phantom experiments based on indocyanine green (ICG), an FDA approved near-infrared (NIR) fluorescent dye, were conducted to evaluate the imaging ability of this device. Two tumor-bearing mice were systematically injected with tumor-targeted NIR fluorescent probes (NIR-830-ATF68-IONP and NIR-830-ZHER2:343-IONP, respectively) and were then imaged to further demonstrate the ability of this FMT probe for imaging small animals.