Contamination of freshwater with nodularin-R (NOD-R) represents a significant global environmental and public health concern. However, ethical problems and technical difficulties surrounding the current detection methods for NOD-R necessitate further studies to devise appropriate alternatives within a regulatory monitoring regime. In this work, we employed an aptamer as a specific recognition element and developed a biolayer interferometry (BLI) biosensor platform for NOD-R detection. The aptasensor we propose displayed a broad detection range from 40 to 600 nM NOD-R (and a linear response range from 40 to 200 nM), and achieved a detection limit as low as 167 pM. In addition, the aptamer-based biosensor was shown to possess high selectivity, as well as good reproducibility and stability. We believe that this novel aptamer-based biosensor provides a potential alternative for the sensitive and rapid detection of NOD-R.