The invention relates to a kit and a detection method for polymorphism detection of a methylenetetrahydrofolate reductase gene. The kit comprises a nucleic acid extraction reagent and a nucleic acid detection reagent, wherein the nucleic acid detection reagent comprises fluorescent probe-containing PCR reaction liquid, a negative control and a positive control; the nucleic acid extraction reagent is a DNA extract; the PCR reaction liquid comprises DNA polymerase, UNG enzyme, a primer, a probe and Mg<2+>; the probe is a molecular beacon probe; and the gene polymorphic site of MTHFR is a C677T polymorphic site. The detection method using the kit is easy and fast to operate, the use of consumables is reduced so as to reduce the detection cost, the detection result is accurate, and the occurrence of false positive result is reduced; by adding the negative control and positive control in the amplification step, the judgment of false positive and false negative can be effectively improved, and the detection precision is higher; since the hairpin structure of the molecular beacon probe is opened with certain force, the specificity to the mutant C677T polymorphic site at the measuring point is higher.