Biodiesel can be produced from vegetable oils, animal fats, and waste cooking oils by transesterification with ethanol (also called ethanolysis) in order to substitute fossil fuels. In this work, the batch ethanolysis of high oleic sunflower oil was transferred into a continuous microstructured device, which induces a better control of heat and mass transfers. Various parameters were studied, notably the initial ethanol to oil molar ratio. An innovative method using NIR spectroscopy was also developed to on-line monitor the transesterification reaction of high oleic sunflower oil with ethanol in microreactors (circular PFA tube 1/1600 OD, 0.0200 ID). The reactions were monitored directly in the microreactors through sequential scans of the reaction medium by the means of an adequate probe. The asset of the method is that no sample collection or preparation is necessary. Partial Least Squares regression was used to develop calibration and prediction models between NIR spectral data and analytical data obtained by a reference method (gas chromatography with flame ionization detection, GC–FID). This method is fast, safe, reliable, nondestructive and inexpensive contrary to conventional procedures, such as gas chromatography and high performance liquid chromatography generally used to determine the composition of crude transesterification medium.