A novel optical biosensor based on simultaneous multiwave length detection surface plasmon resonance (SPR) has been developed for immunosensing. The sensor is designed on the basis of fixing angle of incidence and measuring the reflected intensities of light in the wavelength range of 400–800 nm. The SPR spectra are shown in terms of reflected light intensity verus wavelength of incident light. The intensity of the reflected light is the minimum at the resonant wavelength. The biorecognition surface, formed on a chemically modified gold layer, consists of avidin that is specifically bound with biotin. These sensing membranes were self-assembled on gold layer. The processes of sensing monolayer formation were studied in real time through observing the change of resonant wavelength. The modified surface was used as a model immunosensor and to detect successfully the human factor B (Bf). The Bf was determined in the concentration range of 0.5~100 μg/mL. Under optimum experimental conditions, the sensor has a good repeatability, reversibility and selectivity.