Cell-free gene expression is a vital research tool to study biological systems in defined minimal environments and has promising applications in biotechnology. Developing methods to control DNA templates for cell-free expression will be important for precise regulation of complex biological pathways and use with synthetic cells, particularly using remote, non-damaging stimuli such as visible light. Here, we have synthesised blue light-activatable DNA parts that tightly regulate cell-free RNA and protein synthesis. We found that this blue light-activated DNA could initiate expression orthogonally to our previously generated UV light-activated DNA, which we used to generate a dual-wavelength light-controlled cell-free AND-gate. By encapsulating these orthogonal light-activated DNAs into synthetic cells, we used two overlapping patterns of blue and UV light to provide precise spatiotemporal control over the logic gate. Our blue and UV orthogonal light-activated DNAs will open the door for precise control of cell-free systems in biology and medicine.