A new sensitive, fast thin layer chromatography-fluorescence scanning densitometry(TLC-F) method is described for the simultaneous determination of doxycycline(DOTC), tetracycline(TC) and oxytetracycline(OTC). With a mobile phase chloroform: methanol:acetone: 1% aqueous ammonia(10:22:50:18, V/V) and a silica gel thin-layer, previously treated with 0.27 mol/L sodium EDTA solution adjusted to pH 9.0, all the tetracyclines were separated from each other. The Rf values are 0.52,0.35,0.21 for DOTC, TC and OTC, respectively. The fluorescent spots were scanned with a spectrodensitometer using 365nm as the excitation wavelength and the cut-off filter was set at 440nm. The linear ranges are 0.50∼25.6ng for DOTC; 0.96∼96.2ng for TC and 0.50∼49.6ng for OTC. The relative standard deviations(RSDs) are all <3.5%. The recoveries of them in honey, serum and urine are 94.5%∼107%.