Thyrogen, a recombinant human thyroid-stimulating hormone (rhTSH), has a short half-life in the bloodstream, which necessitates multiple doses during treatment. Therefore, we developed a new long-acting rhTSH using anti-serum albumin Fab-associated (SAFA) technology to validate its biological activity through in vitro assays, pharmacokinetic studies in healthy mice and pharmacodynamics studies in a thyroid-stimulating hormone (TSH)-suppressed mouse model. SAFA-TSH was produced using a Chinese hamster ovary expression system. To verify its biological activity, we generated Nthy-ori 3-1 cells stably overexpressing TSHR and measured the production of cyclic adenosine monophosphate (cAMP). In a rat study, slow-release triiodothyronine (T3) pellets were implanted 3 days before administering Thyrogen or SAFA-TSH to measure the amount of thyroxine (T4) release alone resulting from exogenous administration. SAFA-TSH increased cAMP production dose-dependently, but less effectively than Thyrogen at similar concentrations. SAFA-TSH required six times the dose of Thyrogen to achieve similar cAMP levels, likely due to differences in molecular weight and relative bioactivity. In a rat study, SAFA-TSH produced elevated thyroid hormone levels well after the decline in the response to Thyrogen. SAFA-TSH had significantly higher cumulative effects on T4 and free T4 levels compared with Thyrogen, as observed by a more than two-fold higher average area under the effect curve of 262.56 vs 118.89 μg × h/dL and 127.47 vs 60.75 μg × h/dL, respectively. SAFA technology created successful long-acting TSH that demonstrated bioactivity. These findings endorse the continued development of SAFA-TSH for clinical use, highlighting its potential as a significant advancement treating thyroid cancer patients.