A fluorescent microscopic determination of trace amount of tetracycline is reported based on the aluminum-sensitized fluorescence effect of a self-ordered ring formed by a sessile droplet on glass slide support. Since the evaporative loss of the solvent from the edge wedge of the droplet that is spotted on a hydrophobic-treated glass slide, an outward capillary flow of the interior solvent of the droplet occurs. The resulted outward capillary flow then carries the solute to the perimeter of the droplet spot where the solute accumulates to form a fluorescent self-ordered ring (SOR). Depending on the spotted volume of the aluminum-tetracycline chelate solution, different size of SOR with the outer diameter (o.d.) less than 1.1mm and the ring belt width less than 21.6 microm can be obtained. Data analysis for the imaged SOR by using a digitalized CCD camera showed that the chelate molecule across the fluorescent SOR belt section follows a Gaussian distribution, and the maximum fluorescent intensity (I(max)) was found to be proportional to tetracycline content. When a 0.1 microl droplet was spotted on the solid surface, tetracycline in the range of 7.5-800.0 fmol (or 7.9 x 10(-8) to 800.0 x 10(-8)moll (-1)) can be detected, and the limit of detection can reach 0.8 fmol (or 7.9 x 10(-9) moll (-1)). With present method, the contents of tetracycline in capsule, tablet, urine and fresh milk were satisfactorily detected with the recoveries of 97.0-106.5% and RSD of 1.2-4.2%, correspondingly.