Iodine deficiency results in elevated thyroglobulin (Tg) concentrations, with high iodine Tg being more immunogenic than low iodine Tg. The study investigated the correlation between serum iodine concentration and thyroglobulin autoantibody (TgAb) levels across diverse iodine nutritional statuses as determined by urine iodine concentration (UIC). Demographic information was collected from 1,482 participants through a questionnaire. Blood and spot urine were collected to measure thyroid-stimulating hormone (TSH), TgAb, thyroid anti-peroxidase antibody (TPOAb), serum iodine (SIC), serum non-protein-bound iodine (snPBI), urine iodine (UIC), creatinine (UCr). The median UIC and SIC were 146.5μg/L and 74.9μg/L, respectively. A linear relationship was observed between SIC, snPBI, and serum-protein-bound iodine (sPBI) (P < 0.001). The 90% reference intervals for SIC, snPBI, and sPBI were 50.7-120.7μg/L, 21.9-52.9μg/L, and 19.7-77.9μg/L, respectively. The prevalence of elevated TgAb levels was significantly higher in women than in men (P < 0.001). Both low and high levels of snPBI and sPBI were associated with an increased risk of elevated TgAb levels. In women, the risk of positive TgAb in the group below the reference value of snPBI (OR = 2.079, 95%CI: 1.166, 3.705) and sPBI (OR = 2.578, 95%CI: 1.419, 4.684) was higher. In men, the risk of positive TgAb in the group below the reference value of SIC was higher (OR = 3.395, 95%CI: 1.286, 8.962). Iodine might exert an influence on TgAb levels through its binding to proteins, primarily Tg, thereby altering the iodine content of Tg. The interplay of gender factors further enhanced the risk of TgAb emergence.