Two-photon (TP) microscopy has an advantage for live tissue imaging which allows a deeper tissue penetration up to 1 mm comparing to one-photon (OP) microscopy. While there are several OP fluorescence probes in use for pancreatic islet imaging, TP imaging of selective cells in live islet still remains a challenge. Herein, we report the discovery of first TP live pancreatic islet imaging probe; TP-alpha (Two Photon-alpha) which can selectively stain glucagon secreting alpha cells. Through fluorescent image based screening using three pancreatic cell lines, we discovered TP-alpha from alpha TP fluorescent dye library TPG (TP-Green). In vitro fluorescence test showed that TP-alpha have direct interaction and appear glucagon with a significant fluorescence increase, but not with insulin or other hormones/analytes. Finally, TP-alpha was successfully applied for 3D imaging of live islets by staining alpha cell directly. The newly developed TP-alpha can be a practical tool to evaluate and identify live alpha cells in terms of localization, distribution and availability in the intact islets.