Objective To establish the allele-specific real-time polymerase chain reaction (ASPCR) for detection of neonatal hyperbilirubinemia related gene SLCO1B1 A388G polymorphism and apply this assay to identify the clinical samples.Methods According to SLCO1B1 A388G polymorphism loci,specific primers were designed and the assay was established.Wide type plasmid and mutant plasmid were constructed.Fifty clinical samples were selected,including 30 samples of neonatal hyperbilirubinemia that had been diagnosed with SLCO1B1 A388G mutant and 20 samples of healthy newborns without SLCO1B1 A388G mutant were selected as the controls.Wide type plasmid,mutant plasmid and clinical samples were tested by specific and non-specific primers.A388G polymorphism was determined by difference in Ct (cycle threshold) between specific and non-specific primers.Then,the accuracy,sensitivity and specificity of assay were evaluated.Results The difference in Ct (cycle threshold) between specific and non-specific primers that amplified equivalent wide type template was 13.97 ±0.75.The assay could correctly distinguish the wide type and mutant plasmid.Probit regression analysis showed the sensitivity of the assay could reach to 5.28 copies/μL.For clinical samples,the Ct values of the samples with A388G mutation was less than 37.75 and showed positive results,while the samples without A388G mutation did not show any amplification nor Ct values were larger than 37.75,which showed negative results.Conclusions ASPCR is a fast,simple and effective method for SLCO1B1 A388G polymorphism detection of the clinical simples.It can be used for large sample screening for neonatal hyperbilirubinemia gene loci.