A great challenge of genomics and proteomics is the repeatable and reproducible separation of DNA and proteins with high resolution. Gel electrophoresis is irreplaceably applied for separation and isolation of macromolecules, including DNA, RNA and protein, by providing diffusion resistance to molecules of different size and shape. The separation capability of gel electrophoresis is relatively low for long DNA segment limited by the modest voltage employed for the separation, even for high voltage capillary electrophoresis system. On the other hand, temperature that can affect all physicochemical properties of solution, gel and macromolecules, plays a significant role in gel electrophoresis. Although uncontrolled temperature variation in electrophoresis is considered pestiferous to separation, leading to low reproducibility of separation and thermal degradation of sensitive analytes, a controlled variation in temperature can be beneficial to separation. Temperature has a strong influence on the diffusion coefficient, which determines migration rate in gel electrophoresis. Temperature, at the meantime, affects the structural and mechanical properties of gel such as pore size, gelation rate and elastic modulus, among which the pore size has a significant impact on diffusion. This project describes a photothermal method to enhance gel electrophoretic separation of DNAs. A photothermal temperature gradient is created within an agarose gel using a digital micromirror device (DMD) to dynamically examine the effect of temperature gradient on DNA separation. Temperature gradient between 20-60 °C was established in gel with dynamically controlled patterns. The relation between light brightness and temperature, luminance, and power density was measured for quantitative analysis. While the effect of gel thickness can be ignored in heat transfer.