The EPA has listed thousands of chemicals as potential hormone disruptors, however testing these chemicals is currently a lengthy and expensive process. Here we present a colormetric assay to rapidly detect estrogen or thyroid hormone activity of a sample using cell-free protein synthesis. The biosensor is based on protein folding of an allosteric fusion protein that includes a human thyroid or estrogen receptor. When a chemical binds to the hormone receptor during folding, the protein’s confirmation changes which can then be detected by the reporter enzyme portion of the fusion protein. Using this approach, known hormone disruptors have been rapidly detected including the weakly binding estrogen disruptor BPA. Using lyophilized cell-free reagents, the sensor can be used in a “just-add-sample” format and has potential as a portable at-home assay. We also report the ability of this sensor to function in human blood and urine and the importance of adding RNAse inhibitors for sensing in these mediums.