We report a new SPR (surface plasmon resonance) protein sensor using the Vroman effect for real-time, sensitive and selective detection of protein. The sensor relies on the competitive nature of protein adsorption to the surface, directly depending upon protein’s molecular weight. We, for the first time, utilize the Vroman effect for real-time protein microsensors using three different proteins including annexin (36 kDa), streptavidine (53 kDa), and isolectin (114 kDa) on three different surfaces which are a bare-gold surface and two others modified by OH-and COOH-terminated SAM (Self Assembly Monolayer). The real-time adsorption and displacement of the proteins are monitored by SPR demonstrating very high sensitivity and selectivity label-free protein sensor. The biosensor can distinguish at least 17 kDa in molecular weight with 100 % selectivity. The protein detector can be integrated with microfluidic systems to provide extremely sensitive and selective analytical capability.