In order to definitively diagnosis sporadic Creutzfeldt‑Jakob disease (sCJD), brain tissue is currently required. Therefore, there is a great need for tests that can detect sCJD in body fluids or other types of tissues. Different variables, including the amount of recombinant celluar prion protein (rPrPC), salt, cleaning surfactants and thioflavin T (ThT), in human cerebrospinal fluid (CSF) were evaluated. The reagent concentrations of 1X PBS, 170 mM NaCl, 1 mM EDTA, 0.01 mM ThT and 0.001% SDS, and the amounts of 10 µg rPrPC and 10 µl CSF were considered to be optimal for the real‑time quaking‑induced conversion (RT‑QuIC) assay. Using these conditions, the RT‑QuIC assay for prion protein (PrPSc) detection was observed to be sensitive to 10‑8 diluted brain homogenates of hamsters infected with the 263K scrapie strain. Furthermore, CSF samples from 70 probable sCJD cases and 48 non‑CJD cases were preliminarily screened. A substantial proportion of sCJD samples (57.14%) tested positive by RT‑QuIC, with a short lag phase (<50 h post‑reaction) and high peak ThT values (>25,000 relative fluorescence units). By contrast, only a small number of non‑CJD samples displayed weakly positive results, and these were detected at a later stage (>50 h post‑reaction) and had much lower ThT values. In conclusion, the RT‑QuIC assay in CSF samples reported in the present study may provide a useful pre‑mortem tool for the diagnosis of sCJD, particularly in China where postmortem examination is rarely conducted.