A comparative study was performed to evaluate the signal amplification strategies in electrochemical affinity sensing, which included the direct electron transfer and diffusible-group mediated electron transfer between label enzymes that were specifically bound to target proteins and chemically modified electrode surfaces. As a platform surface for affinity recognition reactions, a double functionalized poly(amido amine) dendrimer monolayer that was modified with ferrocene and biotin groups was constructed on a gold surface. With the chemically modified electrode, a model affinity sensing with avidin was investigated. The advantages of adopting the diffusible-group mediated signaling strategy were demonstrated in terms of signal sensitivity and stability.