In this work, fluorescence spectroscopy was used for the on-line in situ monitoring of the transesterification reaction of soybean oil with ethanol. Different excitation energies in the ultraviolet and visible regions were tested to find the most appropriate excitation wavelength to control the chemical reaction. The excitation wavelength of 532 nm was found to be the most interesting because the luminescence intensity is related to the crude glycerin byproduct, which is associated with the production of alkyl esters. At this excitation wavelength, the oil and biodiesel luminescence signals are minimal. The luminescence signal was monitored in different reactions to investigate its dependence on the temperature, catalyst mass concentration and alcohol: oil molar ratio relation. The methodology used is appropriate to optimize the production process, enabling determination of the best time to stop the reaction as well as the best parameters for the transesterification reaction. (C) 2014 Elsevier Ltd. All rights reserved.