The ion-pair reversed-phase high-performance liquid chromatography (IP-RP-HPLC) of fluorescent-labeled oligonucleotides separation was established, and the concentration of triethylamine-acetic acid (TEAA, 0-0.15 M), pH value (4.5-7.0), and gradient strength were optimized. The retention mechanism of fluorescent dye-labeled oligonucleotides was evaluated by comparing the retention of 5, 10, 15-mer unlabeled oligonucleotides with that of 5'end carboxyfluorescein (5'FAM)-labeled oligonucleotides. In addition, TaqMan TM probes as well as other fluorescent dye-labeled oligonucleotides were considered for separation. The results show that the best resolution of different length fluorescent dye-labeled oligonucleotides was observed at a TEAA concentration of 0.01 M and pH 7.0. The retention behavior of fluorescent-labeled oligonucleotides was significantly different from that of the unlabeled isomers; therefore, they can be separated completely. We found that the retention of unlabeled oligonucleotides enhanced with the increase of the length of the molecule. In contrast, the retention of fluorescent-labeled oligonucleotides reduced with the increase of the length of the molecule. Because the hydrophobicity of fluorescent dyes can greatly impact the retention, the oligonucleotides labeled using fluorescent dyes with higher hydrophobicity would have a longer retention time. However, the effect of the hydrophobicity was limited as the length of the oligonucleotide was increased to a certain level.