Prion diseases are caused by an unconventional infectious agent termed prion, composed mainly of the misfolded prion protein (PrP(Sc)). The development of highly sensitive assays for biochemical detection of PrP(Sc) in blood is a top priority for minimizing the spread of the disease. Here we show that the protein misfolding cyclic amplification (PMCA) technology can be automated and optimized for high-efficiency amplification of PrP(Sc). We show that 140 PMCA cycles leads to a 6,600-fold increase in sensitivity over standard detection methods. Two successive rounds of PMCA cycles resulted in a 10 million-fold increase in sensitivity and a capability to detect as little as 8,000 equivalent molecules of PrP(Sc). Notably, serial PMCA enables detection of PrP(Sc) in blood samples of scrapie-afflicted hamsters with 89% sensitivity and 100% specificity. These findings represent the first time that PrP(Sc) has been detected biochemically in blood, offering promise for developing a noninvasive method for early diagnosis of prion diseases.