A novel cyclopentadienylruthenium(II)thiolato Schiff base complex (Ru(II) thiolato complex), [Ru(SC6H4NC(H)C6H4O(CH2)2SMe)(η5-C2H5)]2, was synthesized and deposited as a self assembled monolayer (SAM) on Au electrode using dichloromethane as the deposition solvent. The SAM’s electroactivity was activated by cycling the electrode in 0.1 M NaOH from 200 mV to +600 mV before using it for electrochemical measurements. Quasi-reversibility of the SAM-modified Au electrode in CH2Cl2 containing tetrabutylammonium tetrafluoroborate (TBATFB) (0.1 M) was confirmed by anodic-to-cathodic CV peak ratio, Ip,a/Ip,c, value of 1.42 and peak separation, ΔEp, value of 85 mV. Randles-Sevčik analysis of the CV data indicated a diffusion limited process. Horseradish peroxidase (HRP) was potentiostatically incorporated on the Au/SAM electrode at +700 mV vs. Ag/AgCl in phosphate buffer saline (PBS) (0.1 M phosphate and 2.7 mM KCl), pH 6.8. The resultant biosensor was used for the amperometric determination of H2O2 in PBS, pH 6.8. at +200 mV vs. Ag/AgCl. The electrode system gave a sensitivity of 5.11 x 10-11 μA mM-1 and a detection limit of 5.26 mM for H2O2.