<p>Immunoassays play a pivotal role in diagnostics due to their high sensitivity and specificity. However, their performance heavily relies on the quality of the antibody used. Conventional full-length antibodies can be limited by factors such as stability under demanding conditions, relatively high production costs, and engineering complexities related to their size and structure. These limitations may restrict their utility in certain diagnostic scenarios. Nanobodies, with their unique structural and physicochemical properties, have emerged as ideal next-generation diagnostic reagents. Their superior target penetrability, flexibility in biological modification, and cost-effective expression capabilities effectively address challenges in immunoassays, including steric hindrance and interference from complex matrices. We systematically summarize the preparation workflows for nanobodies, with a focus on nanobody-based immunoassays platforms such as enzyme linked immunosorbent assay (ELISA), lateral flow immunochromatography (LFIA), electrochemical/optical sensors, and microfluidic chips. Furthermore, we summarize the applications of these platforms against cancer biomarkers, bacterial/viral pathogens, and inflammatory factors. Specifically, we provide an in-depth discussion and comparison of the strengths and limitations of nanobodies within each platform, along with effective strategies to enhance performance. Collectively, this review offers novel insights and practical guidelines for nanobody-driven immunoassays, highlighting their potential to improve current diagnostic methods.</p>