Abstract: To address the growing demand for accurate calibration of isothermal polymerase chain reaction (PCR) analyzers in the market, this study developed calibration reagents and a standard system for isothermal PCR analyzers based on loop-mediated isothermal amplification (LAMP) technology. Using the specific values and characteristic sequences from fluorescence quantitative PCR instrument standard materials (GBW(E)091100-091108) as the reference, LAMP regular primers (F3, B3, FIP, BIP) and loop primers (LF, LB) were designed. The optimal regular and loop primers, along with the ideal reaction temperature, were identified through screening tests. The performance of the calibration kit was assessed by evaluating the concentration gradient range of the corresponding standard materials, as well as the kit's sensitivity and reproducibility. Calibration reagent performance was further validated using commercially available isothermal PCR analyzers. Results indicated that the optimal reaction temperature for the LAMP calibration kit is 65℃, with the standard material concentration range covering seven orders of magnitude (10⁰ to 10⁶ copies/μL) and a sensitivity threshold of 10⁰ copies/μL. The kit also demonstrated excellent repeatability. The developed LAMP-based calibration reagents are suitable for the calibration of isothermal PCR analyzers, supporting their performance evaluation and market regulation. This research lays a solid foundation for the development and implementation of national calibration and verification standards for isothermal PCR analyzers.