Controlled immobilization of proteins is crucial in many applications, including biosensors. Allergen biosensing, for example, requires molecular recognition of suitably immobilized proteins by specific antibodies and sensitive measurement of this interaction. Self-assembled monolayers (SAMs), terminated by active functions, and are of great interest for the immobilization of biomolecules. The efficiency of further biorecognition involving molecules immobilized on these surfaces demonstrates an interesting dependence on the chain length and terminal function of the SAM. This motivated us to investigate adsorption of two proteins both known as milk allergens-β-lactoglobulin and apo-transferrin-on amine-terminated SAMs. We varied the chain length by using either short or long chain amine-terminated thiols (cysteamine, CEA, and 11-mercaptoundecylamine, MUAM). We also investigated the influence of the addition of a rigid cross-linker, p-phenylene diisothiocyanate (PDITC), to these amine layers prior to protein adsorption. Protein binding was studied using polarization modulation-infrared reflection absorption spectroscopy (PM-IRRAS) and atomic force microscopy (AFM) to characterize their amount and dispersion. We found that protein immobilization varies with SAM chain length and is also influenced by the presence of a cross-linker. The presence of a rigid cross-linker favours the binding of proteins on long chain SAMs, while the effect is almost nonexistent on shorter chains. In addition, the presence of the cross-linker induces a better dispersion of the proteins on the surfaces, regardless of the length of the thiols forming the SAMs. The effects of chain length and chemistry of protein binding are discussed.