The invention discloses a primer pair, a probe and a kit used for detecting polymorphism of an MTHFR gene. The kit comprises: the primer with the base sequences represented by SEQ ID No.1 and SEQ ID No.2, which is designed for the c.677 site of the MTHFR gene, wherein the first base at the 5' end of the base sequence represented by the SEQ ID No.1 has an amino group modification group, and the first base at the 5' end of the base sequence represented by the SEQ ID No.2 has an amino group modification group; and the probe with the base sequences represented by SEQ ID No.3 and SEQ ID No.4, which is designed for the c.677 site of the MTHFR gene, wherein one base of the base sequence represented by the SEQ ID No.3 has an amino group modification group, and one base of the base sequence represented by the SEQ ID No.4 has an amino group modification group. Compared with kits in the prior art, the kit disclosed in the invention has the advantages of remarkable shortening of the detection time, reduction of nonspecific amplification, detection of SNPs and point mutation, reduction of the design difficulty and the detection cost, no influences on the diagnosis precision, realization of mutation detection using a single reaction tube without cover opening, maximal avoiding of aerosol pollution, convenience in clinic use, and reduction of operation induced errors.