Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes COVID-19, has resulted in an ongoing global pandemic that has already claimed more than 4 million lives. While most antiviral efforts have focused on essential SARS-CoV-2 proteins, RNA structural elements within the viral genome are also compelling targets. In this study, we identified high-affinity L-DNA aptamers against a SARS-CoV-2 stem-loop II-like motif (s2m), a highly conserved RNA structure with promising diagnostic and therapeutic potential. Optimized L-C1t and L-C3t aptamers bind selectively to s2m RNA with K_d values in the nanomolar range, and are capable of differentiating the monomeric s2m stem-loop from the proposed homodimer duplex. This structure-specific mode of recognition also allows L-C1t and L-C3t to discriminate between s2m RNAs from SARS-CoV-2 and SARS-CoV-1, which differ by just two nucleotides. Finally, we show that L-C1t and L-C3t induce dramatic conformational changes in s2m structure upon binding, and thus, have the potential to block protein–s2m interactions. Overall, these results demonstrate the feasibility of targeting SARS-CoV-2 RNA using L-aptamers, which has important implications in the diagnosis and treatment of COVID-19. Moreover, the high affinity and selectivity of L-C1t and L-C3t, coupled with the intrinsic nuclease resistance of L-DNA, present an opportunity for generating new tools and probes for interrogating s2m function in SARS-CoV-2 and related viruses.