Modern diagnostic technologies rely on both in vitro and in vivo modalities to provide a complete understanding of the clinical state of a patient. Nanoparticle-antibody conjugates have emerged as promising systems to confer increased sensitivity and accuracy for in vitro diagnostics (e.g., immunoassays). Meanwhile, in vivo applications have benefited from the targeting ability of nanoparticle-antibody conjugates, as well as payload flexibility and tailored biodistribution. This review provides an encompassing overview of nanoparticle-antibody conjugates, from chemistry to applications in medical immunoassays and tumor imaging, highlighting the underlying principles and unique features of relevant preclinical applications employing commonly used imaging modalities (e.g., optical/photoacoustics, positron-emission tomography, magnetic resonance imaging, X-ray computed tomography).