The spectroscopic ellipsometry (SE), and attenuated internal reflection spectroscopic ellipsometry (TIRE) are promising methods in label-free biosensing applications. An ellipsometer running under surface plasmon resonance (SPR) conditions has unique advantages over other SPR-based methods in terms of sensitivity and real-time/label-free measurement capability. In this study, both SE and TIRE-based brevetoxin B (BTX) sensors were developed using two anti-BTX aptamers reported before. A new aptamer sequence was also derived from these two antiBTX aptamers using predictive modeling tools and an exclusion method. All three antiBTX aptamers' analytical performances were quite competitive in terms of both detecting range and detection limits. However, the selectivity of the previously reported aptamers against analogs of BTX was poor at low detection ranges, especially for okadaic acid. Furthermore, the selectivity of the derived aptamer was lower than its predecessors. The sensors were capable of detecting BTX in the range of 0.05 nM–1600 nM in the TIRE and 0.5 nM–2000 nM in the SE configuration. The detection limits of the sensors were 1.48 nM (1.32 ng/mL) and 0.80 nM (0.72 ng/mL) for SE and TIRE configurations, respectively. Both configurations have been used successfully to detect BTX standards spiked into real fish and shrimp samples.