Transforming growth factor-β (TGF-β) regulates multiple fundamental physiological processes and is closely related to severe diseases such as cancer, fibrosis, immune disorders and cardiovascular diseases. TGF-β is thus an important biomarker for clinical diagnosis and prognosis, and a crucial target for therapeutics development. Here we describe a high-content, serum-free, easy-to-use, and cost-effective (CAGA)-EGFP cell-based biosensor for accurate measurements of active TGF-β. Together with non-destructive and continuous measurement protocol and data processing method established here, the biosensor is capable of detecting active TGF-β1 in the range of 0.024-6.25 ng/mL concentration with >91% accuracy and high repeatability. Overall, the engineered (CAGA)-EGFP biosensor is a powerful tool for detection of active TGF-β and for mechanistic study of the TGF-β pathway. The greatly reduced cost and operating simplicity also makes it a highly potent in vitro platform for high-throughput screening of anti-TGF-β therapeutics.