A competitive enzyme-linked immunosorbent assay (ELISA) with generated mouse anti-consensus interferon (CIFN) antibody was developed for quantitative determination of pegylated consensus interferon (PEG-CIFN) in rhesus monkey serum. The operational concentrations of the original assay were determined using the chessboard method. ELISA working range was 10-5000 ng/ml, corresponding to a limit of quantification of 8.4 ng/ml in rhesus monkey serum. In a precision test, intra-assay CV ranged 1.8-9.6% and inter-assay CV ranged 3.5-12.7%. Relative recovery rate of this ELISA assay ranged from 102.65-115.77%, with RSD values ranging from 2.26-5.44%. Three groups of rhesus monkeys received 1250 microg/kg, 300 microg/kg, 150 microg/kg PEG-CIFN by subcutaneous administration, and blood samples were drawn via the femoral vein at the specified time points. PEG-CIFN in rhesus monkey serum was determined using the competitive ELISA, and the results were compared with antiviral activity assay. In conclusion, the competitive ELISA assay we developed has sufficient sensitivity, precision, and accuracy for the analysis of a rhesus monkey serum sample.