Measuring low amounts of anti-erythropoietin antibodies (anti-EPO Abs) is important to evaluate the therapeutic safety of recombinant human erythropoietin (rhEPO). In this work, a simple, sensitive and high-throughput chemiluminescent (CL) imaging assay was developed for the detection of anti-EPO Abs in human sera. The influence of several physicochemical parameters, such as coating conditions, incubation time, detergent concentration and exposure time, were investigated. A calibration curve was established and the range of quantitative detection was 0.12-13.91 ng/mL. The limit of detection (LOD, 3sigma) for the CL-imaging assay was 0.033 ng/mL. Compared to conventional colorimetric enzyme-linked immunosorbent assay (ELISA), the LOD of the CL-imaging assay is 50-fold lower. The recoveries of anti-EPO Abs in the fortified serum were in the range 87.1-116.9% using the present method, which highlighted the validity of the CL-imaging assay system to accurately determine the anti-EPO Abs in serum samples. CL-imaging assay was used to evaluate the presence of anti-EPO Abs in serum samples obtained from chronic renal failure (CRF) patients treated with rhEPO. Contrary to what was expected, the sera from CRF patients did not contain anti-EPO Abs.