Due to their intrinsic nuclease resistance, L-oligonucleotides are being increasingly utilized in the development of molecular tools and sensors. Yet, it remains challenging to synthesize long L-oligonucleotides, potential limiting future applications. Herein, we report straightforward and versitile approach to assemble long L-RNAs from two or more shorter fragments using T4 RNA ligase 1. We show that this approach is compatible with the assembly of several classes of functional L-RNA, which we highlight by generating a 124 nt L-RNA biosensor that functions in serum.