Background The global pandemic of coronavirus disease 2019 (COVID-19) has led to the development of multiple detection kits by national manufacturers for severe acute respiratory syndrome coronavirus 2 viral nucleic acid testing. The purpose of this study is to evaluate the performance of different kits (i.e., Maccura kit and Sansure kit) in real clinical work using clinical samples, which will help with the optimization of the test kits. Method During the past three months (March–May 2022), 1399 pharyngeal swabs from suspected COVID-19 patients have been initially screened using the Maccura kit in Jilin, China, and the test results were verified using the Sansure kit. The cycle threshold (Ct) values generated by the two kits were compared at different viral load levels. Correlation and consistency of the Ct values were investigated using Spearman correlation, Deming regression, and Bland–Altman plots. The cut-off Ct values of the Maccura kit were recalculated by referencing the result of the Sansure kit as a standard. Furthermore, another 163 pharyngeal swabs from suspected COVID-19 patients were collected to verify the new cut-off values. Results As a result of the Maccura kit testing, 1192 positive cases and 207 suspected COVID-19 cases were verified. After re-examination by the Sansure kit, 1118 positive cases were confirmed. The difference between the Ct values provided by the two kits was statistically significant, except for the N gene at high viral load. The Ct values obtained from the two kits presented a linear positive correlation. The Maccura kit used new cut-off Ct values of 35.00 (ORF1ab gene) and 35.07 (N gene). Based on that, the validation pass rate for the new cut-off Ct values was 91.41%. Conclusion Since the Maccura kit is found to have false positives in actual clinical work, recalculation of the cut-off values can reduce this occurrence. In order to improve the accuracy of the testing, laboratories should use two kits for COVID-19 testing, and the adjusting and optimizing of the kits for their situation are needed.