In this work, the Repetitive cyclic voltammetry method used to electrodeposition of thin Ruthenium film from the ionic liquid (bmimPF6) solution of RuCl3. The FESEM, EDX, XPS, and AFM used to evaluate and characterization of prepared film. The imaging results indicated well-regulated nanoclusters (RuNCs) of ruthenium. The electrochemical behavior of prepared nanocluster film was investigated by using cyclic voltammetry. The electrodeposited nanofilm shows excellent electrocatalytic activity toward enzyme less oxidation of glucose in the natural pH. The deposited film enhances the electron transfer efficiency and reduces the oxidation potential to next to −0.1 V vs. Ag/AgCl. The quantitative determination of glucose without any interference at RuNCs/ ITO has obtained in a width dynamic range of 20 μM–1.2 mM with a limit of detection of 20 μM. It could be a good candidate for generating miniaturized glucose biosensor in humane matrices.