A new competitive bead immunoassay (CBIA) based on Luminex technology for detecting clenbuterol in urine was reported. The carboxylated fluorescent beads were directly coated with clenbuterol derivatives without carrier protein spacer. Clenbuterol antibody was biotinylated, which was used for clenbuterol detection in combination with the functionalized bead and streptavidin-phycoerythrin (SAPE). The effects of spacer on the CBIA method were investigated. The results indicated that the presence of small molecular spacer between bead and hapten improved the assay sensitivity and the hydrophilic spacer (glycine) was better than the hydrophobic spacer (m-aminobenzoic acid) for this CBIA method. The study affirms the importance of the judicious choice of hapten derivatives in the CBIA method for detecting small molecule drug based on Luminex technology. The method could be used for clenbuterol detection in livestock urine and possible for the simultaneous detection of multiple veterinary drugs.