Development of real-time sensor based on the target-specific probe that make possible sensitive, rapid and selective detection and monitoring of the particular antigen molecules could be of substantial importance to the many applications. Because of its high specificity to the target molecules, excellent temperature stability, and easy production, bacterial phage might serve as a powerful biorecognition probe in biosensor applications. Here, we report extremely sensitive and specific label-free direct detection of model antigen, beta-galactosidase (beta-gal), based on surface plasmon resonance (SPR) spectroscopy. The beta-gal specific landscape phage 1G40 has been immobilized on the gold surface of SPR SPREETA sensor chip through physical adsorption [V. Nanduri, A.M. Samoylova, V.Petrenko, V. Vodyanoy and A.L.Simonian, Comparison of optical and acoustic wave phage biosensors, 206th Meeting of The Electrochemical Society, Honolulu, Hawaii, October 3-8, (2004)]. Another non-specific to the beta-gal phage, a wild-type phage F8-5, was used in the reference channel. The concentration-dependent binding of beta-gal in both channels were assessed by monitoring the sensor optical response as a function of time under different experimental conditions, and the concentration of beta-gal was computed in differential mode. Concentrations of beta-gal between 10(-12) M and 10(-7) M could be readily detected, with linear part of calibration curve between 10(-9) M and 10(-6) M. When beta-gal was pre-incubated with different concentrations of free 1G40 phage prior to exposure to the biosensor, concentration-dependent inhibition was observed, indicating on biosensor high specificity toward beta-gal. Apart from a flow through mode used to deliver the samples to the surface for the SPR sensor, batch mode sensing was also employed to study the binding of beta-gal to immobilized phage on the SPR sensor surface. Experiments using a flow through mode provided more consistent results in the full dose range and showed higher sensitivity as opposed to the batch mode studies. The mean K(d) and binding valences for the flow through mode studies was 1.3+/-0.001 nM and 1.5+/-0.03, in comparison to 26+/-0.003 nM and 2.4+/-0.01 for the batch mode studies. The average thickness of phage 1G40 adlayer deposited through flow through and batch mode was 3+/-0.002 and 0.66+/-0.001 nm, respectively.