Single nucleotide detection is important for early detection of diseases and for DNA sequencing. Monolayer (ML) MoS₂ nanopores have been used to identify and distinguish single nucleotides with good signal-to-noise ratio in the recent past. Here, we use a bilayer (BL) MoS₂ nanopore (∼1.3 nm thick) to detect distinct single nucleotides with high spatial resolution and longer dwell time. In this study, the performance of similar sized (<3 nm) ML and BL MoS₂ nanopores for detection of a single nucleotide has been compared. Both single nucleotide and single stranded DNA translocations through them are studied. For single nucleotide detection, we observe that BL MoS₂ nanopores demonstrate twice the dwell time as compared to ML MoS₂ nanopores with 95% confidence. Single nucleotide detection rate for BL MoS₂ nanopores (50–60 nucleotides per s) is five-fold higher as compared to ML MoS₂ nanopores (10–15 nucleotides per s) in 10 pM analyte concentration. For single stranded DNA, we observe 89% (for 60 DNA molecules detected) single nucleotide detection efficiency with BL MoS₂ nanopores as compared to 85% for ML MoS₂. The DNA sequencing efficiency through BL MoS₂ nanopores is also found to be 8–10% better than through ML MoS₂ nanopores, irrespective of DNA sequencing orientation. Thus, owing to improved analyte/nanopore charge interaction BL MoS₂ nanopores can be used for single nucleotide detection with high resolution due to longer dwell time, detection rate and efficiency. This study demonstrates the improved ability of BL MoS₂ nanopores in sequencing DNA with 8–10% higher efficiency, two-times temporally resolved single-nucleotide current signatures and five-times higher detection rate, compared to ML MoS₂ nanopores.