The bioluminescent protein Gaussia luciferase (GLuc) was fused to an anti-carcinoembryonic antigen (CEA) antibody fragment, the diabody, for in vivo optical tumor imaging. A 15-amino acid N-terminal truncation (GLΔ15) resulted in a brighter protein. Fusions of the anti-CEA diabody to full-length GLuc and GLΔ15 retained high affinity for the antigen, emitted light, and exhibited excellent enzymatic stability. In vivo optical imaging of tumor-bearing mice demonstrated specific targeting of diabody-GLΔ15 to CEA-positive xenografts, with a tumor/background ratio of 3.8 ± 0.4 at four hours after tail-vein injection, compared to antigen-negative tumors at 1.3 ± 0.1 (p = 0.001). MicroPET imaging using ¹²⁴I-diabody-GLΔ15 demonstrated specific uptake in the CEA-positive tumor (2.6% ID [injected dose]/g) compared to the CEA-negative tumor (0.4% ID/g) at 21 hours. Although further optimization of this fusion protein may be needed to improve in vivo performance, the diabody-GLΔ15 is a promising optical imaging probe for tumor detection in vivo.