An direct competitive immunofluorescence method was developed in plate to detect chlortetracycline residues in environment using the Lumichrome as fluorescent probe. Different reaction conditions such as pH , the concentration of antigen and antibody and incubation times were assayed. Under optimized conditions, chlortetracycline can be determined in the concentration range of 1×10-8--2×10-4 mol/L, and the linear equation was y=11.363x+93.026, R2=0.99. The detection limit of this method IC90 was 3.74×10-7mol/L. The recoveries of chlortetracycline at various spiking levels in the sludge samples ranged from 95.43% to 104.26%, with relative standard deviations of 3% to 6%. These data demonstrate that the excellent performance of the proposed method in the detection of chlortetracycline in water samples.