Objective An aptamer specifically binding to diethyl thiophosphate (DETP) was constructed and incorporated in an optical sensor and electrochemical techniques to enable the specific measurement of DETP as a metabolite and a biomarker of organophosphate exposure. Results A DETP-bound aptamer was selected from the library using capillary electrophoresis-systematic evolution of ligands by exponential enrichment (CE-SELEX). A colorimetric method revealed that the aptamer had the highest affinity for DETP, with a mean K-d value (+/- SD) of 0.103 +/- 0.014 mu M. The docking results and changes in resistance showed that the selectivity of the aptamer for DETP was higher than that for the similar structures of dithiophosphate (DEDTP) and diethyl phosphate (DEP). The altered amplitude of cyclic voltammetry showed a linear range of DETP detection covering 0.0001-10 mu g/ml with a limit of detection of 0.007 mu g/ml. The recovery value of a real sample of pH 7 was 97.2%. Conclusions The current method showed great promise in using the DETP-specific aptamer to detect the exposure history to organophosphates by measuring their metabolites, although degradation of organophosphate parent compounds might occur.