Humic substances (HS), especially humic acids (HA), interact with several pollutants or xenobiotics, as organic (e.g., drugs, agrochemicals, and petroleum derivatives) and inorganic (e.g., toxic metals) chemical substances. Oxytetracycline (OTC) is a broad-spectrum tetracycline antibiotic applied to animal and human infections. Despite its large use in livestock production, it can promote antibiotic resistance, which is a global concern. This Chapter describes the fluorescence study addressed to the molecular interaction between OTC antibiotic and a HA extracted from a Brazilian tropical soil, in order to understand the absence or presence of this interaction and the influence of organic matter (OM) components on the environmental mobility of OTC. Furthermore, the physical fundamentals of spectroscopy are presented and explored. Thus, the presence of molecular interaction between OTC and HA was observed, which was strongly dependent on the pH value of the medium.