A novel biosensor based on a two-dimensional gradient (TDG) guided-mode resonance (GMR) filter was introduced in this study. The TDG-GMR is demarcated in terms of the gradient grating period (GGP) in one dimension and gradient waveguide thickness (GWT) in the other dimension. A single compact sensor can combine these two features to simultaneously provide a broad detection range through GGP and high resolution through GWT. A detection range of 0.109 RIU (0%–60% sucrose content) with a limit of detection of 5.62 × 10−4 was demonstrated in this study by using a TDG-GMR with a size of 140.8 × 125.4 µm2. This value cannot be achieved using one dimensional gradient GMR sensor. Label-free (LF) biomolecule detection through TDG-GMR was also experimentally demonstrated in a model assay of albumin. The result confirms that the GWT-GMR provides a better resolution, whereas the GGP-GMR provides a broader detection range. A device for multiplex measurement could be easily implemented with a compact sensor chip and a simple readout directly from a charge-coupled device. This system would require a narrow-band source such as a light emitting diode or a laser diode, in addition to a limited number of other components such as a polarizer and a collimator. The proposed TDG-GMR could easily be integrated with smartphones and portable devices.