Abstract: Addressing the challenges in the calibration process of nucleic acid extractors, this study explores the key metrological characteristics and measurement traceability system based on the JJF 1874-2020 standard, "Calibration Specification for (Automatic) Nucleic Acid Extractors." Using common nucleic acid extractors as examples, this paper analyzes the calibration methods and uncertainty assessments for four metrological characteristics: temperature, vibration frequency, and nucleic acid extraction efficiency. The results include temperature indication errors of 0.5 ℃ at 55 ℃ and 0.7 ℃ at 70 ℃, temperature uniformity of 0.3 ℃ at 55 ℃ and 0.5 ℃ at 70 ℃, temperature stability of ±0.1 ℃, vibration frequency stability of ±0.1 Hz, nucleic acid extraction efficiency of 82.9%, efficiency homogeneity of 6.3%, and repeatability of 4.5%. The study evaluates the uncertainty of temperature indication errors and nucleic acid extraction efficiency. Compared to the JJF 1874-2020 standard, additional uncertainty components from the micro spectrophotometer, pipettor, and electronic balance are included. The study summarizes the types and pros and cons of temperature and vibration frequency measuring devices, analyzes factors affecting nucleic acid recovery rate, and highlights the importance of using consistent extraction methods and reagents. It proposes solutions and optimization methods for calibration challenges and discusses the concept and detection methods of cross-contamination rates in nucleic acid extractors, with an experimental finding of 0.0% cross-contamination rate.