In this work, we developed a method to quantify ethanol in ethyl soybean biodiesel and ethyl biodiesel from waste frying oil using mid-infrared spectroscopy in association with multivariate calibration by the partial least-squares (PLS) method. The obtained models are efficient in ethanol determination in concentrations ranging from 0.14% to 1.00% (w/w). In both PLS models low values of the root-mean-square error prediction (0.02%, w/w) and excellent correlation between measured and predicted values in the prediction set (R > 0.99) were observed, and there were no systematic errors according to the ASTM E1655 standard. The methods were validated according to international and national guidelines by the estimate of figures of merit, such as accuracy, linearity, sensitivity, selectivity, analytical sensitivity, and limits of detection and quantification. Considering these good results, the proposed method can be used for biofuel quality control in a fast, simple, and nondestructive way.