Abstract: Objective: To establish a rapid detection method for human methylene tetrahydrofolate reductase (MTHFR) gene polymorphism by using the primer mismatching amplification and fluorescence quantitative PCR. Methods: A total of 214 samples with different MTHFR C677T genotypes (CC, CT, TT) or different A1 298C genotypes (AA, AC, CC), which were verified by gene sequencing, were collected, and the plasmids with the corresponding wild-type and homozygous mutants were constructed, respectively. The amplification refractory mutation system (ARMS) primers and TaqMan probes were designed based on the wild-type standard sequence of MTHFR gene, and the optimal mutation detection system was established. The results from the system were compared with the known sequencing results to verify the feasibility of the system. Results: The performance of the established TaqMan-ARMS method was excellent, which had 10 copies/μL of lowest detectable limit and high specificity. There was no nucleic acid amplification in the cross detection between samples and the negative control. In addition, the established method had good repeatability. The standard deviations of the reproducibility detection of MTHFR-667 and 1 298 loci ranged from 0.11 to 0.44, and the coefficients of variation (CV) of homozygous and heterozygous samples were all less than 4.52%. The consistency of the established method with the sequencing method in 214 clinical samples was 100%. Conclusion: The established TaqMan-ARMS method for the detection of MTHFR gene polymorphism is simple, rapid and accurate, which may be used for the rapid diagnosis of clinical patients.