Here we present a validated rapid detection system for propofol, an anaesthetic with a narrow therapeutic window, in whole blood. This method utilises an on-line molecularly imprinted polymer solid-phase extraction, rather than the traditional C18 solid-phase extraction, coupled to fluorescence optical fibre detection. The linearity was assessed from 0.10–15 μg mL⁻¹ of propofol in whole blood, and the coefficients were greater than 0.995. The absolute recoveries of propofol were 95.81, 97.56 and 97.93% at three different concentrations. The inter-batch precision ranged from 4.3% to 8.1%, and the accuracy value ranged from 102.5% to 104.4%. The developed method was successfully applied to measure propofol concentrations in simulated whole blood samples. The entire analysis procedure lasted only 5 minutes, and the results showed no statistical difference between the new on-line method and a validated high-performance liquid chromatography method. The new on-line method, however, is faster and more convenient for the clinical real-time detection of propofol than previously reported methods.