Human epidermal growth factor receptor 2 (Her2) is abundantly expressed in various solid tumors. The Her2specific Affibody (Z Her2:2891 ) has been clinically tested in patients with Her2-positive breast cancer and is regarded as an ideal drug carrier for tumor diagnosis and targeted treatment. Indocyanine green (ICG) can be used as a photosensitizer for photothermal therapy (PTT), in addition to fluorescent dyes for tumor imaging. In this study, a dimeric Her2-specific Affibody (Z Her2 ) based on Z Her2:2891 was prepared using the E. coli expression system and then coupled to ICG through an N-hydroxysuccinimide (NHS) ester reactive group to construct a novel bifunctional protein drug (named ICG-Z Her2 ) for tumor diagnosis and PTT. In vitro, ICG-Z Her2mediated PTT selectively and efficiently killed Her2-positive BT-474 and SKOV-3 tumor cells rather than Her2-negative HeLa tumor cells. In vivo, ICG-Z Her2 specifically accumulated in Her2-positive SKOV-3 tumor grafts rather than Her2-negative HeLa tumor grafts; high-contrast tumor optical images were obtained. However, Her2-negative HeLa tumor grafts were not detected. More importantly, ICG-Z Her2 -mediated PTT exhibited a significantly enhanced antitumor effect in mice bearing SKOV-3 tumor grafts owing to the good photothermal properties of ICG-Z Her2 . Of note, ICG-Z Her2 did not exhibit acute toxicity in mice during short-term treatment. Overall, our findings indicate that ICG-Z Her2 is a promising bifunctional drug for Her2-positive tumor diagnosis and PTT.