A sensitive chemiluminescence (CL) method, based on the inhibitory effect of roxithromycin (ROX) on the CL reaction between luminol and dissolved oxygen in a flow-injection system, was first proposed for the determination of ROX at picogram levels. The decrement of CL intensity was linearly proportional to the logarithm of ROX concentrations ranging from 0.1 to 100 pg mL-1, giving the limit of detection (LOD) of 0.03 pg mL-1 (3σ). At a flow rate of 2.0 mL min-1, a complete analytical procedure including sampling and washing could be performed within 0.5 min, with relative standard deviations (RSDs) of less than 5.0% (n=5). The proposed procedure was applied successfully to the determination of ROX in pharmaceutical, human serum, and urine with the recoveries ranging from 90.0 to 110.0%.