Objective:To establish mono-tube allele-specific amplification for typing multiplex SNPs simultaneously. Methods:Taking three SNPs of 71G > A,425A > T,and 758G > A in the TYR gene as example,a PCR pre-amplification was firstly carried out for producing a long target containing all SNPs of interest. Secondly,the preamplified DNA fragments were digested by a restriction endonuclease to form sticky ends,which were then ligated to a designed DNA adapter by ligase. Thirdly,an allele-specific amplification was performed by using all allele-specific primers and a universal primer in a single tube by using the adapter-ligated DNA as templates. Finally,the allele-specific amplification products were separated by agarose gel electrophoresis,and the types of the SNPs were discriminated by the length of the amplified products. Results:Three SNPs in the TYR gene were successfully typed for 30 healthy Mainland Chinese and the results were in agreement with those obtained by RFLP. Conclusion:This method with high specificity is accurate and cost-effective,and can be used for typing multiple SNPs simultaneously.