Abstract Three different kinds of glassy carbon (GC-R, GC-K, GC-G) were equally pretreated, further modified with electrochemically deposited Prussian Blue and used as sensors for hydrogen peroxide at an applied potential of À 50 mV (vs. Ag j AgCl). Their performance was evaluated with respect to the following parameters: the coverage and electrochemistry of the electrodeposited Prussian Blue, the sensitivity and the lower limit of detection for hydrogen peroxide, and the operational stability of the sensors. GC-R showed the best behavior concerning the surface coverage and the operational stability of the electrodeposited Prussian Blue. For this electrode the sensitivity for hydrogen peroxide (10 mM) was 0.25 A/M cm 2 and the detection limit was 0.1 mM. Scanning electron microscopy was used to study the surfaces of the three electrodes before and after the electrodeposition of Prussian Blue and to search for the reason for the three different behaviors between the different glassy carbon materials. The Prussian Blue modified GC-R was also used for the construction of a glucose biosensor based on immobilizing glucose oxidase in Nafion membranes on top of electrodeposited Prussian Blue layer. The operational stability of the glucose biosensors was studied in the flow injection mode at an applied potential of À 50 mV (vs. Ag j AgCl) and alternatively injecting standard solutions of hydrogen peroxide (10 mM) and glucose (1 mM) for 3 h. For the GC-R based biosensor a 2.8% decrease of the initial glucose response was observed