The direct interaction of hairpin DNA with 9-hydroxyfluorene (9-OHFLU) through hydrogen bonds was investigated by electrochemical impedance spectroscopy (EIS), UV-Vis spectroscopy and ¹H NMR spectra. Based on these results, an electrochemical hairpin DNA sensor was developed for the detection of 9-OHFLU by EIS. Upon 9-OHFLU interacting with hairpin DNA film on the gold electrodes, the charge transfer resistance (R_CT) of the hairpin DNA film was significantly increased and remained constant after 30 min. Depending on the difference in charge transfer resistance (ΔR_CT) before and after 9-OHFLU interaction with the hairpin DNA, 9-OHFLU could be detected with a concentration as low as 1 nM. However, only a much smaller ΔR_CT appeared when eight selected hydroxyl polycyclic aromatic hydrocarbons (HO-PAHs) interacted with the hairpin DNA for 30 min, which demonstrated that 9-OHFLU could be discriminated from other HO-PAHs by EIS. The performance of the sensor in real lake water sample was further explored for the detection of 9-OHFLU with the detection limit of 4 nM.