For the assessment of the dynamic concentration of exogenous protein or peptide in serum, an antibody- independent sandwich system based on the avidin-biotin interaction was established and validated. The exogenous protein or polypeptide labeled with biotin was added to the microplate coated with streptavidin, and followed by adding HRP-streptavidin to complete the sandwich system. This sandwich system was validated with respect to specificity, sensitivity, accuracy (recovery) and reproducibility. While the sensitivity of detection could reach to 0.3125 μg/L, the sensitivity and the range of detection can also be adjusted by changing coating concentration of streptavidin. Recoveries ranged from 97.82% to 107.29%, and the intra- and inter-assay variation was < 5.76% and < 8.42%, respectively. Thus, the well-validated streptavidin sandwich system was successfully applied to determine dynamic concentrations of Biotin-HAS (human serum albumin) and Biotin-OVM (Ovomucoid) in mouse serum, respectively. Most importantly, this novel method, where generation of antibody or radionuclide is not needed, may provide a new choice for assessment or monitoring of exogenous protein or peptide in pharmacokinetics study.