To eliminate the influence of electrochemically active compounds on ethanol determination in fruit juices via biosensor, an ethanol-assisted enzyme electrode was prepared and a novel alcohol biological detection system was constructed using the synergy of working and auxiliary enzyme electrodes. The anti-interference property of the system was tested and the anti-interference coefficient was fitted. Thereafter, the calculation formula of ethanol concentration was deduced to achieve the rapid and accurate detection of ethanol concentration. The results showed that the anti-interference performance of the detection was stable with good stability, accurate results, and strong ability. When the method was applied to determine the ethanol concentration in fruit juices, the relativity standard division was 0.80% and the recoveries were 99.10% to 101.10%. Glucose, sucrose, citric acid, Ca²⁺, catalase, and vatamine C in the fruit juices had no effect on ethanol detection. T-test analysis showed that there was no significant difference between this method and the national standard and gas chromatography (P>0.05).The method is simple in operation,low in cost and high in accuracy,which can meet the production requirements of low cost.