Background Determination of UGT1A1 (TA)(n) polymorphism prior to irinotecan therapy is necessary to avoid severe adverse drug effects. Thus, accurate and reliable genotyping methods for (TA)(n) polymorphism are highly desired. Here, we present a new method for polymerase chain reaction (PCR) melting curve analysis using one fluorescent probe to discriminate the UGT1A1*1 [(TA)(6)] and *28 [(TA)(7)] genotypes. Methods After protocol optimization, this technique was applied for genotyping of 64 patients (including 23 with UGT1A1*1/*1, 22 with *1/*28, and 19 with *28/*28) recruited between 2016 and 2021 in China-Japan Friendship Hospital. The accuracy of the method was evaluated by comparing the results with those of direct sequencing and fragment analysis. The intra- and inter-run precision of the melting temperatures (T(m)s) were calculated to assess the reliability, and the limit of detection was examined to assess the sensitivity. Results All genotypes were correctly identified with the new method, and its accuracy was higher than that of fragment analysis. The intra- and inter-run coefficients of variation for the T(m)s were both <= 0.27%, with standard deviations <= 0.14 degrees C. The limit of detection was 0.2 ng of input genomic DNA. Conclusion The developed PCR melting curve analysis using one fluorescent probe can provide accurate, reliable, rapid, simple, and low-cost detection of UGT1A1 (TA)(n) polymorphism, and its use can be easily generalized in clinical laboratories with a fluorescent PCR platform.