AbstractWith the goal of developing a quencher‐free probe composed of an artificial nucleic acid, the fluorescent nucleobase analogue 5‐(perylenylethynyl)uracil (PeU), which was incorporated into totally artificial serinol nucleic acid (SNA) as a substitute for thymine, has been synthesized. In the context of a 12‐mer duplex with RNA, these fluorophores reduce duplex stability slightly compared with that of an SNA without PeU modification; thus suggesting that structural distortion is not induced by the modification. If two PeUs were incorporated at separate positions in an SNA, the fluorescent emission at λ≈490 nm was clearly enhanced upon hybridization with complementary RNA. A quencher‐free SNA linear probe containing three PeUs, each separated by six nucleobases, has been designed. Detection of target RNA with high sensitivity and discrimination of a single‐base mismatch has also been demonstrated.