A biocompatible, cost-effective, and scalable reduced graphene oxide (rGO) film was obtained from shellac using thermal treatment and its structural, chemical, and electrical properties were investigated. This thermally-decomposed rGO (TrGO) film exhibited good crystallinity, low sheet resistance, and high carbon content. TrGO flakes obtained from the film were dispersed and drop cast onto indium tin oxide/glass electrodes to fabricate label-free electrochemical immunosensors for the quantitative detection of the influenza virus H1N1 via electrochemical impedance spectroscopy. These sensors exhibited high stability and reproducibility, both possibly ascribable to the high adhesion of TrGO due to its phenolic-OH moiety; the limits of detection were 26 and 33 plaque-forming units, respectively, in phosphate-buffered saline and diluted saliva. These cost-effective TrGO-based sensors showed great potential as reliable and robust nanomaterial-based biosensors for widespread clinical applications.