Cell-free gene expression is a vital research tool to study biological systems in defined minimal environments and has applications in biotechnology. Developing methods to control DNA templates for cell-free expression will be important for precise regulation of complex biological pathways and drug delivery, particularly using remote, non-damaging stimuli such as visible light. Here, we have synthesised blue light-activatable DNA parts to selectively control cell-free expression. Using this approach, we were able to control cell-free RNA and protein expression with blue light. Due to the orthogonality of this blue light-activated DNA to UV irradiation, we combined it with our previously generated UV light-activated DNA to generate a dual-wavelength controlled cell-free AND-gate. This blue light-activated DNA was also used to control synthetic cells; lipid-bounded compartments that mimic cellular function. Our blue and UV orthogonal light-activated DNAs will open the door for precise control of cell-free systems in biology and medicine.