The immunosensor has been proven a versatile tool to detect various analytes, such as food contaminants, pathogenic bacteria, antibiotics and biomarkers related to cancer. To fabricate robust and reproducible immunosensors with high sensitivity, the covalent immobilization of immunoglobulins (IgGs) in a site-specific manner contributes to better performance. Instead of the random IgG orientations result from the direct yet non-selective immobilization techniques, this review for the first time introduces the advances of stepwise yet site-selective conjugation strategies to give better biosensing efficiency. Noncovalently adsorbing IgGs is the first but decisive step to interact specifically with the Fc fragment, then following covalent conjugate can fix this uniform and antigens-favorable orientation irreversibly. In this review, we first categorized this stepwise strategy into two parts based on the different noncovalent interactions, namely adhesive layer-mediated interaction onto homofunctional support and layer-free interaction onto heterofunctional support (which displays several different functionalities on its surface that are capable to interact with IgGs). Further, the influence of ligands characteristics (synthesis strategies, spacer requirements and matrices selection) on the heterofunctional support has also been discussed. Finally, conclusions and future perspectives for the real-world application of stepwise covalent conjugation are discussed. This review provides more insights into the fabrication of high-efficiency immunosensor, and special attention has been devoted to the well-orientation of full-length IgGs onto the sensing platform.