Amplification/hybridization-based genetic analyses using primers containing locked nucleic acids (LNAs) present many benefits. Here, we developed a novel design for universal fluorescent PCR using LNAs. Universal fluorescent PCR generates intermediate nonlabeled fragments and final fluorescent fragments in a two-step amplification process that uses locus-specific primers with universal tails and universal fluorescent primers. In this study, a few standard nucleotides were replaced with LNAs only in the fluorescent universal primers. The sequence of the fluorescent universal primer significantly affected the amplification efficiency. For primers with three LNAs, the fluorescent primers with stable M13(-47) sequences provided the most efficient signal (approximately tenfold higher than the primers with M13(-21) sequences at lower Tm values). Moreover, AT-rich LNA substitutions in the fluorescent primers produced much lower amplification efficiencies than GC-rich substitutions. GC-rich LNAs produced greater differences in Tm values among primers, and resulted in the preferential production of fluorescently labeled amplicons. The specificity and sensitivity of LNA-containing fluorescent primers were assessed by genotyping eight STRs in Japanese individuals, and full STR profiles could be generated using as little as 0.25 ng of genomic DNA. The method permitted clear discrimination of alleles and represents sensitive STR genotyping at a reduced cost.