Immuno-PET/CT imaging, a branch of molecular imaging, can noninvasively and specifically visualize biomarker expression across the body. Trophoblast cell surface antigen 2 (Trop2) is a pan-cancer biomarker and plays a crucial role in tumorigenesis through multiple signaling pathways. The study aims to develop and translate novel Trop2 single-domain antibody (sdAb) tracers for clinical use. Methods: Two sdAbs (i.e., His-tagged T4 and His-tag−free RT4) are recombinantly expressed in Chinese hamster ovary cells. The purities and binding kinetics are determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis, high-performance liquid chromatography, and surface plasmon resonance assays. The AlF restrained complexing agent (RESCA) method is applied to develop ¹⁸F-labeled sdAb tracers ([¹⁸F]AlF-RESCA-T4 and [¹⁸F]AlF-RESCA-RT4), followed by thorough preclinical imaging and blocking studies on tumor-bearing mice and a pilot clinical trial evaluating the clinical imaging safety and feasibility of [¹⁸F]AlF-RESCA-T4 immuno-PET/CT. Results: [¹⁸F]AlF-RESCA-T4 and [¹⁸F]AlF-RESCA-RT4 possess high radiochemical purities. Preclinical imaging in the T3M-4 tumor model revealed prominent uptake (percentage injected dose/g) of [¹⁸F]AlF-RESCA-T4 (11.13 ± 1.53, n = 4) and [¹⁸F]AlF-RESCA-RT4 (8.83 ± 1.22, n = 4), which were significantly reduced by coinjection of unlabeled T4 and RT4 in blocking studies. The His-tag removal strategy further optimized the probe’s in vivo pharmacokinetics and reduced renal radioactivity accumulation without significantly decreasing tumor uptake. In a pilot clinical trial, [¹⁸F]AlF-RESCA-T4 immuno-PET/CT showed promising potency in annotating Trop2 expression and differentiating tumors from inflammatory diseases such as tuberculosis. Conclusion: [¹⁸F]AlF-RESCA-T4 and [¹⁸F]AlF-RESCA-RT4 can specifically annotate Trop2 expression. Clinical [¹⁸F]AlF-RESCA-T4 immuno-PET/CT imaging can screen patients for Trop2-targeted therapies and differentiate lung inflammation from cancer.