Naphthenic acids (NAs) are water-soluble components of petroleum. The characterization and quantification of NAs by analytical methods have proved quite challenging, whilst the toxic effects of these water-soluble compounds on a variety of organisms adversely affecting reproduction and steroid production is becoming apparent. In this study, we report a fluorescence-based competitive binding method for rapid sensing of the presence of NAs using cellulosic peptide array strips as sensors. The peptide array was designed from sequences derived from the estrogen receptor (ER). Several of these peptides were able to detect the presence of NAs at low micromolar (∼5 mg L−1) levels in different oil sands process affected water samples. The specific binding of one of the peptides, peptide 17 (EGXVEIFDXLLATS) with NAs was evaluated using isothermal titration calorimetry. The results show that peptide 17 interacts strongly with NAs with an apparent binding constant (Ka) of 96 × 106 M−1, and may bind NAs in a similar fashion as ER interacts with estrogen. Finally, the data support that the peptides displaying high affinity for NAs can be used for developing disposable peptide-based sensor arrays for NA detection in oil sands process affected water samples