In this study, the development of biosensors capable of bi-enzyme reactions by including Horseradish peroxidase and glucose oxidase was carried out for detection of glucose. The sensors were manufactured using electro deposition method to reduce production time, and screen printed electrodes (SPE) were used to produce economical sensors. To check the bienzyme effect, the sensor was compared and analyzed with single enzyme biosensor. The characteristics of the sensor were evaluated using scanning electron microscopy(SEM), cyclic voltammetry( CV), electrochemical impedance spectroscopy(EIS), chronoamperometry(CA), and flow injection analysis(FIA). Analysis results from SEM, CV and EIS confirmed that the enzymes are well fixed to the electrode surface. In addition, it was confirmed that bi-enzyme biosensors manufactured from the CA method improved signal performance by 200% compared to single enzyme biosensors. From this results, we were able to explain that HRP and GOD react catalyzed to each other. And the results of FIA showed that the intensity of each current signal was constant when the same concentration of glucose was injected four times. In addition, by analyzing the intensity of current signals for glucose concentrations, the biosensors manufactured in this study showed excellent trends in signal sensitivity, reproducibility and stability.