Many chemical mediators regulate neutrophil recruitment to inflammatory sites. Although the actions of each of these chemical mediators have been demonstrated with neutrophils in vitro, how they act cooperatively or counteract each other in vivo remains largely unknown. To understand the behaviors of neutrophils in vivo, the activities of intracellular signaling molecules must be visualized in living tissues. For this purpose, we can use genetically-encoded biosensors based on the principle of Förster resonance energy transfer (FRET). In this review, we first provide an overview of FRET biosensors and then describe how we can utilize these biosensors to visualize the activity changes of signaling molecules in neutrophils during extravasation. In relation to this topic, we will also describe the development of transgenic mice expressing the FRET biosensors and in vivo two-photon excitation microscopy.