Detection of autoantibody against extractable nuclear antigens (ENAs) plays a critical role in the diagnosis and management of autoimmune diseases.In this study, we assessed the performance of LETIA in detecting anti-ENAs. Total 606 serum samples from the Second Hospital of Shanxi Medical University were collected. Anti-SSA, anti-SSB, anti-Sm, anti-U1-snRNP, and anti-Sm/RNP were parallelly detected by LETIA and line immunoblot (LIA). Besides, this study assessed LETIA for its repeatability in detecting anti-ENAs autoantibodies, and consistency with LIA. A receiver operating characteristic (ROC) curves was drawn to assess the accuracy of LETIA. The LETIA and LIA showed high coincidence rate in detecting anti-SSA, anti-SSB, anti-Sm, anti-U1-snRNP, and anti-Sm/RNP autoantibodies, with the results being 87.22%, 96.61%, 97.03%, 88.28%, and 92.06%, respectively. Almost perfect consistency (kappa > 0.8) were found in the detection of anti-SSB and anti-Sm by LETIA and LIA. While in the detection of anti-SSA, anti-U1-snRNP, and anti-Sm/RNP, moderate consistency (0.6 ⩽ kappa ⩽ 0.8) were shown. The AUCs of anti-SSA, anti-SSB, anti-Sm, anti-U1-snRNP, and anti-Sm/RNP detected by LETIA were 0.972 (95% confidence interval (CI): 0.941–1.000, p < 0.001), 0.986 (95% CI: 0.967–1.000, p < 0.001), 0.912 (95% CI: 0.763–1.000, p < 0.001), 0.829 (95% CI: 0.731–0.928, p < 0.001), and 0.828 (95% CI: 0.715–0.941, p < 0.001), respectively. LETIA and LIA showed considerable consistency in detecting anti-ENAs. Moreover, with the pronounced advantages of automatic and rapid detection, and high universality, LETIA can meet the requirements for quantitative detection of anti-ENAs.