Monitoring biologically active substances have been an important one to know the impact of these substances on the environment. The development of techniques that seek to degrade or mitigate the toxicity of these substances has been of great interest. Thus, simple, low cost and accurate analytical procedures are essential for monitoring and controlling these techniques. In this work, the analytical procedure was proposed to determine the antibiotic sulfanilamide with spectrofluorimetric detection in river water samples. The procedure is based on radiation emission at 365 nm when excited at 255 nm, in acetic acid medium 0.10 mol/L. The procedure presented linearity between 1.14 to 11.2 μg mL−1 and a quantification limit of 1.4 μg mL. Sulfanilamide determination can be performed with a recovery ranging from 90.3 to 106%. The accuracy of determining a solution of 1.4 μg/l was 1.5% (n=10). The spectrofluorimetric procedure for determination and sulfanilamide is simple, fast (30 samples per hour), and does not require complex sample preparation.