We describe the redox functionalization of graphene oxide (GO) and the development of versatile amperometric biosensing platforms for clinically important analytes such as cholesterol ester, uric acid and glucose. Ferrocene (Fc) redox units were covalently tethered onto the GO backbone using diamine sigma spacers of different chain lengths (C3-, C6-, and C9-diamines). The functionalized GO (Fc-GO) displays a pair of redox peak corresponding to Fc/ Fc+ redox couple at ∼0.225 V. The surface coverage and heterogeneous electron transfer rate constant of Fc-GO depends on the length of sigma spacer. Amperometric biosensors for cholesterol (total), uric acid and glucose have been developed by integrating Fc-GO and the respective redox enzymes with screen printed electrode. Fc-GO efficiently mediates the bioelectrocatalytic oxidation of the substrates in the presence of the redox enzymes. The spacer length of Fc-GO controls the bioelectrocatalytic response of the biosensing platforms. The sensitivity of the biosensors based on C9 sigma spacer is significantly higher than the others. The detection limit (S/N = 3) of the biosensors for cholesterol and uric acid was 0.1 μM and for glucose it was 1 μM. Excellent stability, reproducibility, selectivity and fast response time were achieved. Biosensing of cholesterol, uric acid and glucose in human serum sample is successfully demonstrated with the biosensors, and the results are validated with the clinical laboratory measurement.