Rapid and simple detection of single nucleotide polymorphism (SNP) is vital for individualized diagnosis and eventual treatment in the current clinical setting. In this study, we developed a tetra-primer ARMS-PCR combined lateral flow assay (T-ARMS-PCR-LFA) method for simultaneous visual detection of two alleles. By using four primers labeled with digoxin, biotin and Cy5 separately in one PCR reaction, the amplified allele-specific products could be captured by streptavidin and the anti-Cy5 antibody on two separated test lines of a LFA strip, which allows the presentation of both alleles within the single LFA strip. Both DNA and whole blood can be used as templates in this genotyping method in which the whole detection process is completed within 75 minutes. The performance assay of T-ARMS-PCR-LFA demonstrates the accuracy, specificity and sensitivity of this method. One hundred human whole blood samples were used for MTHFR C677T genotyping in T-ARMS-PCR-LFA. The concordance rate of the results detected was up to 100% when compared with that of the sequencing results. Collectively, this newly developed method is highly applicable for SNP screening in clinical practices.