A newly sensitive TLC-fluorescence scanning densitometer (TLC-F) is described for the simultaneous determination of tetracycline (TC) and 4-epimeric tetracycline (4-ETC), using a mobile phase system of chloroform: methanol:acetone:1% aqueous ammonia (10:22:53:15, v/v) and a silica gel thin-layer, previously treated with 0.27mol/L sodium EDTA solution adjusted to pH 9.0. The TC and 4-ETC were well separated and their Rfvalues are 0.64 and 0.24, respectively. The excitation wavelength was set at 365nm. The linear ranges are 0.96˜96.2 ng for TC and 0.96˜48.1 ng for 4-ETC. The limit of detection for TC and ETC are 0.31ng and 0.57ng, respectively. The epimeric reaction between TC and 4-ETC was studied. Their recoveries in urine are 97%∼106% and the relative standard deviations (RSDs) of both are less than 4.0%.