<p>Silk fibroin (SF) is a natural biopolymer extracted from <i>Bombyx mori</i> that has emerged as a promising sustainable material in biosensing applications, especially because of its biodegradable and tunable properties. However, SF is still insufficiently explored in electrochemical biosensing. We present a novel <i>all-in-one</i> electrochemical immunoplatform based on a disposable electrode surface sequentially modified with Prussian Blue (PB) and SF. First, PB is electrodeposited, acting as a redox mediator that enhances signal amplification through its reversible electrochemistry and its low reduction potential for H₂O₂. The SF layer is here presented as a novel, biocompatible matrix for antibody immobilization that offers structural versatility and sustainability. This dual modification generates a synergistic ecofriendly functional interface that is stable at room temperature. Onto this innovative surface, we have developed a sandwich-type electrochemical immunosensor employing a biotinylated detector antibody and streptavidin–horseradish peroxidase (Strep-HRP) as the enzymatic label for the determination of T-cell immunoglobulin and mucin-domain containing protein 1 (TIM-1, also known as KIM-1 or HAVCR1), a relevant biomarker associated with cancer-related angiogenesis and poor prognosis, and, more recently, with allergenic episodes. The platform enabled sensitive and selective determination of TIM-1 in plasma samples from colorectal cancer (CRC) patients, and saliva samples from individuals undergoing allergic episodes, in which elevated TIM-1 levels were observed. A remarkable advantage of this immunosensor is its operational stability for up to 20 days at room temperature, eliminating the need for a cold chain.</p> Graphical Abstract <p></p>