Upon subjecting molecules to nonequilibrium conditions, many biophysical and biochemical features such as molecular diffusion, protein folding, dissociation constant, as well as enzyme-catalyzed reactions can be characterized in an aqueous solution. However, conducting assays under nonequilibrium conditions in complex self-assembled biomatrices (e.g., extracellular matrices) remains challenging due to the limitations associated with sample handling, reaction design, and optical detection. Herein, we present the investigation of biomolecular thermodiffusion in noncovalently assembled synthetic or naturally derived hydrogels. This approach has been demonstrated with a large variety of analytes of different sizes across the nanoscale, including small molecules, polysaccharides, proteins, DNA, and five DNA origamis of different geometries in various polymer networks. As the aggregation of analytes can be suppressed, the in-biomatrix method has also shown advantages over in-solution measurements. Remarkably, the method provides a unique opportunity to study how a thermophoretic movement of matrix surroundings can impact the thermophoretic movement of analytes, with dimensions from low to high nm and a million-fold variation in mass. Most importantly, the method is capable of measuring binding affinity in biomatrices, allowing for characterizing the protein-ligand interaction within a more biologically relevant context.