Optical techniques for visualization and quantification of chemical and biological analytes are always highly desirable. Here we show a hyperspectral surface plasmon resonance microscopy (HSPRM) system that uses a hyperspectral microscope to analyze the selected area of SPR image produced by a prism-based spectral SPR sensor. The HSPRM system enables monochromatic and polychromatic SPR imaging and single-pixel spectral SPR sensing, as well as two-dimensional quantification of thin films with the measured resonance-wavelength images. We performed pixel-by-pixel calibration of the incident angle to remove pixel-to-pixel differences in SPR sensitivity, and demonstrated the HSPRM’s capabilities by using it to quantify monolayer graphene thickness distribution, inhomogeneous protein adsorption and single-cell adhesion. The HSPRM system has a wide spectral range from 400 nm to 1000 nm, an optional field of view from 0.884 mm2 to 0.003 mm2 and a high lateral resolution of 1.2 μm, demonstrating an innovative breakthrough in SPR sensor technology.