There is a request of high throughput measurement when surface plasmon resonance (SPR) biosensors are applied to real-time measure binding reaction of bio-molecular, so SPR biosensors need wide-field imaging ability. A differential interferometry SPR imaging biosensor based on Mach-Zehnder configuration which can decrease environment noise effectively is set up. An algorithm is created to extract phase information in differential phase-sensitive SPR imaging biosensor. After signal and reference beams are combined, p and s polarization beams are splitted by a Wollaston prism and received by CCD after focused by lens. A correlation algorithm is used to align these two light spots. The phase is extracted from several periods of p and s polarization interference intensity changes and the noises decreases simultaneously by the average. The differential phase is calculated by point to point subtraction. The differential phase image is obtained by scanning in this way. For phase information is extracted from time axis in the system, there isn't decrease exist in spatial resolution. The system detects one data per 10 s. Spatial resolution of the system is 31.25 μm×20.83 μm and refractive index resolution is 10-5RIU.