Silanization is one of the widely explored surface modification strategies for bio‐functionalization of oxide interfaces. For biosensor applications, silanes with active terminal groups such as amine, thiol, carboxylic, and aldehyde groups are utilized in routine. In near‐field sensing schemes like biologically sensitive field‐effect transistors (BioFETs), it is crucial to generate a homogenous layer of silane to confine the bio‐interactions in close vicinity of the sensor interface. The homogeneity of such biofunctional layer is determined by the surface activation and silanization protocol being applied. Here, the impact of surface activation process and silanization on electrical characteristic of field‐effect devices was studied comprehensively using an electrolyte‐oxide‐semiconductor (EOS) capacitor with a high quality gate oxide. The thermally grown silicon oxide (SiO2) interface was activated using acidic mixtures and plasma treatment, while the subsequent silanization steps were investigated comparatively using two different silanes (3‐aminopropyl triethoxysilane (APTES) and 3‐glycidyloxypropyl trimethoxysilane (GPTMS) in wet‐chemical and vapour‐phase processes. Furthermore, the optimized silanization process was utilized to immobilize an oligo strand at the EOS capacitor surface, followed by the hybridization of complementary oligo strands. The optimized protocol holds potential for large‐scale production of functional oxide interfaces for various applications.This article is protected by copyright. All rights reserved.