Bifunctional hydroxyl/thiol-functionalized fourth-generation polyamidoamine dendrimer (G4-PAMAM)-encapsulated Au nanoparticle (NP) was synthesized and immobilized on a mixed self-assembled monolayer (SAM)-modified gold surface. This modified surface was resistant to nonspecific adsorption of proteins having a wide range of molecular weight and isoelectric points. Part of the dendrimer thiol groups were converted to hydrazide functionalities providing an activated surface available to subsequently immobilize the receptor for developing a sensor surface to immunoaffinity reaction. Herein, the surface plasmon resonance (SPR) detection of insulin was obtained by means of a competitive immunoassay principle. The resulting Au NP dendrimer-modified surface provided an assay with high stability, significantly enhanced sensitivity, and a detection limit for analyzing insulin of 0.5 pM. The SPR detection of insulin was amplified due to the changes in the dielectric properties of the matrixes, occurring upon the biorecognition processes on the sensor surface, through the coupling of the localized plasmon of the NPs with the surface plasmon wave. The developed sensor chip was used to analyze insulin in human serum samples from healthy and diabetic patients. The results showed good correlation to the reference method. The specificity and the improved sensitivity of this biosensing platform could have significant implications for the detection of a wide range of molecules and biomarkers in complex biological media.