Background: Radioactive ¹³¹I (RAI) therapy is used in patients with differentiated thyroid cancer (DTC) after total thyroidectomy for remnant ablation, adjuvant treatment or treatment of persistent disease. ¹³¹I retention data, which are used to indicate the time at which a ¹³¹I treated DTC patient can be released from the hospital, may bring some insights regarding clinical factors that prolong the length of hospitalization. The aim of this study was to investigate the ¹³¹I whole-body retention in DTC patients during ¹³¹I therapy. Methods: We monitored 166 DTC patients to follow the ¹³¹I whole-body retention during ¹³¹I therapy with a radioactivity detector fixed on the ceiling of each protected room. A linear regression fit permitted us to estimate the whole-body ¹³¹I effective half-life in each patient, and a relationship was sought between patients’ clinical characteristics and whole-body effective ¹³¹I half-life. Results: The effective ¹³¹I half-life ranged from 4.08 to 56.4 h. At multivariable analysis, longer effective ¹³¹I half-life was related to older age and extensive extra-thyroid disease. Conclusions: ¹³¹I effective half-life during ¹³¹I treatment in DTC patients is highly variable among patients and is significantly longer in older and in patients with RAI uptake in large thyroid remnants or in extrathyroidal disease that significantly prolongs the whole-body retention of ¹³¹I.