This work describes an immunochemical approach for the quality control of Paeoniae Radix by an enzyme-linked immunosorbent assay (ELISA) based on determination of the total concentration of paeoniflorin (PF) and albiflorin (Alb), which are major bioactive constituents in Paeoniae Radix. Four hybridromas secreting monoclonal antibodies against PF and Alb were produced by fusing splenocytes from a mouse immunized by a PF-bovine serum albumin (BSA) conjugate with the hypoxanthine-aminopterin-thymidine (HAT)-sensitive mouse myeloma cell line, P3-X63-Ag8-653. A relatively higher reactivity of monoclonal antibodies (MAbs) with PF and Alb than oxypaeoniflorin (OP) and benzoylpaeoniflorin (BP) was observed, while other monoterpenes and benzoic acid did not cross-react. When PF was used as a standard, the assay can cover a measuring range of 20—600 ng/ml for PF and Alb. A series of Paeoniae Radix samples have been determined, and the results showed good agreement with that determined by traditional high-performance liquid chromatography (HPLC). The developed competitive ELISA was 100 times more sensitive than the HPLC method. Meanwhile, fifteen Chinese traditional prescriptions were determined by the competitive ELISA.