A simple and selective spectrofluorimetric method for the detection of chlortetracycline (CTC) was studied. In pH 7.4 buffer medium l-tryptophan (l-Trp), applied as the fluorescence probe, interacted with CTC resulting in fluorescence quenching of the probe. CTC was detected with maximum excitation and emission wavelengths at λex /λem = 275/350 nm. Notably, quenching of fluorescence intensities was positively proportional to the CTC concentration over the range of 0.65-30 μmol L-1 and the limit of detection was 0.2 μmol L-1 . Effect of temperature shown in Stern-Volmer plots, absorption spectra and fluorescence lifetime determination, indicated that fluorescence quenching of l-Trp by CTC was mainly by static quenching. The proposed study used practical samples analysis satisfactorily.