Five hybridoma cell lines secreting anti-PrP antibodies were established from the fusion between mouse myeloma Sp2/0 and spleen cells from mice immunized with recombinant Chinese Luxi yellow cattle (Bos taurus, Luxi) PrP (24-234) or recombinant Chinese small-tailed Han sheep PrP (94-227). According to their Western blot reactivity, five monoclonal antibodies (mAbs) could be divided into two groups. Group A, mAbs 1H2, 4C6, and 4C11 recognized re-PrP, PrP(C), and PrP(Sc) from both bovine and sheep. Group B, mAbs 2H3 and 4H10 only recognized re-PrP and PrP(Sc) of sheep, and especially, these two mAbs could not recognize PrP(C) from both bovine and sheep. In immunohistochemistry (IHC) test, mAb 4C11 immunostained the PrP(Sc) accumulation in tissue sections from BSE cattle and Scrapie sheep, and compared with mAb 6H4, it had the same immunohistochemical pattern. An IHC method based on mAb 4C11 for the detection of BSE was established and had been applied for the long-term surveillance of BSE in China. From 2001 to 2004, 12,692 samples from the whole country had been tested and all had negative results.