Many diseases and disorders are linked to exposure to endocrine disrupting chemicals (EDCs) that mimic the function of natural estrogen hormones. Here we present a Rapid Adaptable Portable In-vitro Detection biosensor platform (RAPID) for detecting chemicals that interact with the human estrogen receptor β (hERβ). This biosensor consists of an allosteric fusion protein, which is expressed using cell-free protein synthesis technology and is directly assayed by a colorimetric response. The resultant biosensor successfully detected known EDCs of hERβ (BPA, E2, and DPN) at similar or better detection range than an analogous cell-based biosensor, but in a fraction of time. We also engineered cell-free protein synthesis reactions with RNAse inhibitors to increase production yields in the presence of human blood and urine. The RAPID biosensor successfully detects EDCs in these human samples in the presence of RNAse inhibitors. Engineered cell-free protein synthesis facilitates the use of protein biosensors in complex sample matrices without cumbersome protein purification. Display omitted •Cell-free protein synthesis (CFPS) detection of estrogenic endocrine disruptors•CFPS in blood and urine improved with addition of RNAse inhibitors.•Rapid CFPS detection of the E2 in human blood and urine