Effective drugged driving screening by the roadside requires simple-to-use sensing platforms that allow accurate measurement of drugs from biofluids. Achieving this goal is of vital importance to prevent and control the incidence of drug abuse, traffic accidents and crime. Given this, we designed a rapid, sensitive and reagentless electrochemical aptamer-based (EAB) sensor to detect ketamine. This platform produced nanomolar detection accuracy and reliable results were obtained within 30 s. To design a sensitive platform, circular dichroism (CD) was used to screen the aptamer which underwent the largest binding-induced conformational change. In addition, kinetic differential measurement (KDM) was utilized to improve the signal gain of the EAB sensor. The constructed sensing platform achieved sensitive and specific detection of ketamine in 50 % saliva and undiluted urine with detection limits as low as 10 nM, which was much lower than the physiological detection threshold. Hence the present EAB sensor is expected to be applied in the preliminary drugged driving screening in the real world.