Fine-needle aspiration biopsy (FNAB) is a routine diagnostic test for thyroid nodules. The use of local anesthesia (LA) before the procedure is still controversial. This prospective study aimed to evaluate the degree of pain and specimen adequacy in liquid-based cytology (LBC) for FNAB of thyroid nodules with and without LA. A total of 100 consecutive patients with thyroid nodules who underwent FNAB with and without LA between January and December 2020 were included. Patients who received LA had a significantly lower immediate pain scale score (P = 0.01). Multivariate analysis demonstrated that no use of LA (odds ratio [OR] = 3.48, 95% confidence interval [CI] = 1.50–8.10, P = 0.004) and lesion abutting the trachea (OR = 6.14, 95% CI = 1.56–24.12, P = 0.009) were independently and significantly associated with pain degree immediately after FNAB. A higher proportion of patients who previously underwent FNAB thought that LA was helpful and should be performed prior to FNAB. However, the use of LA did not improve the specimen adequacy (P = 0.075). The results showed that administration of LA with a proper technique before ultrasound-guided FNAB might reduce immediate pain after the procedure, and patients may experience more pain when the aspirated nodules abut the trachea.