A novel continuous flow biosensor based on gold nanoparticles and poly(propylene-co-imidazole) was developed for the online determination of p-benzoquinone. The amperometric response was measured as a function of p-benzoquinone concentration at an applied potential of À 50 mV. The hydrogen peroxide concentration was optimized and fixed at 1 mM in samples. The mass transfer resistance of the copolymer film was minimized, and the flow cell was regenerated quickly at 1 mL/min. The resulting device provided good analytical performance based on a linear dynamic range from 5-100 µM, a short response time of 3 s, a detection limit of 3.3 µM, excellent repeatability with a relative standard deviation of 0.82%, long-term stability of 95% after four weeks, and an accuracy of 105%. The gold nanoparticles enhanced the electron transfer rate on the electrode. The apparent Michaelis-Menten constant was 4 mM, showing that the enzyme retained catalytic specificity and provided high activity for p-benzoquinone.