Cell-free expression of a gene to protein has become a vital tool applied in DNA nanodevices and synthetic cells. Antisense oligonucleotides are often used to induce gene knockdown; however, they have been underexplored for cell-free applications. Developing methods to non-invasively control gene knockdown with antisense oligonucleotides will be crucial for their precise regulation of cell-free biology and biotechnology. Here, we report a mild method for selectively attaching photoremovable protecting groups, photocages, onto phosphorothioate linkages of antisense oligonucleotides. Using this photocaging method, upon illumination, the original phosphorothioate antisense oligonucleotide is reformed. Photocaged antisense oligonucleotides, containing mixed phosphorothioate and phosphate backbones, showed a drastic reduction in duplex formation and RNAse H activity, which was recovered upon illumination. For the first time, we demonstrated that photocaged antisense oligonucleotides can be used to control cell-free protein synthesis. This technology will have future applications in light-activated biological logic gates and controlling the activity of synthetic cells.