Biotinylated denatured bovine serum albumin (Bt-dBSA)-coated cadmium telluride (CdTe) quantum dot (QD) conjugates were prepared and used to develop the multiplexed fluoroimmunoassay for the simultaneous determination of five chemical residues. An immune complex was formed using avidin as the bridge to link the Bt-dBSA-QDs with the antibodies. Primarily, individual quantitative determinations of five representative chemical residues were carried out based on the different emission properties of the QDs. Five antibodies were then conjugated with the corresponding QDs to establish the indirect competition fluorescent-linked immunosorbent assay (ic-FLISA) for the simultaneous detection of five chemicals in one well of a microplate. The linear range for dexamethason (DEX) was from 0.33 microg/kg to 10 microg/kg, 0.28 microg/kg to 10 microg/kg for gentamicin (GM), 0.16 microg/kg to 25 microg/kg for clonazepam (CZP), 0.17 microg/kg to 10 microg/kg for medroxyprogesterone acetate (MPA) and 0.32 microg/kg to 25 microg/kg for ceftiofur (CEF), respectively. The limit of detection (LOD) for the simultaneous determination of DEX, GM, CZP, MPA and CEF were as low as 0.13 microg/kg, 0.16 microg/kg, 0.07 microg/kg, 0.06 microg/kg and 0.14 microg/kg, respectively. This detection method was used to analyze samples of pork muscle and the recoveries ranged from 61.3% to 80.3% for DEX and from 74.0% to 87.2% for MPA. Further more, good correlation between the novel ic-FLISA and traditional ELISA was demonstrated during the determination of DEX and MPA residues in real samples. The QD-based protocol described here is less time consuming than the classical method and it may be sufficiently flexible to be used in other systems for the simultaneous multicolor detection of the drugs.