This study is a debut of a side-by-side comparison of different methods to modify the surfaces of the silica-based substrates for biosensor fabrication. The fabricated biosensors are assessed for the quantification of cardiac troponin I (cTnI), a biomarker of acute myocardial infarction, at ultralow concentrations and small increment resolution in human serum. To accomplish this, silica samples and silicon nanowire field-effect transistor (SiNWFET) channels were modified with (3-aminopropyl)triethoxysilane, 1-(3-aminopropyl)silatrane, and the mixed self-assembly monolayer of silane-polyethylene glycol (silane-PEG, constituting of silane-PEG-NH2 and silane-PEG-OH at the ratio of NH2:OH = 1:10). Afterwards, they were treated with glutaraldehyde to immobilize aptamer probes for cTnI aptasensor detection. Thereafter, the modified-silica samples were investigated for surface roughness and antifouling capability, whereas the SiNWFET aptasensors were employed to determine cTnI. Empirical data revealed that only the silane-PEG-modified samples provided a superior surface for probe immobilization and high antifouling capability for the PEG-SiNWFET aptasensors to determine cTnI at ultralow levels with high resolution in human serum. Therefore, surface modification with silane-PEG provided an option for the challenge of precise quantification of cTnI, as well as of other protein biomarkers, at ultralow levels in bio-samples using the FET-based biosensors.