Biodiesel is an evolution toward the attempt to substitute fossil fuel by renewable biomass. Typically, blends of diesel and biodiesel are commercialized instead of pure biodiesel for economic and technical reasons. It would be of interest for customers, regulations agencies, and dealers regarding custody transfer of biodiesel blends to have a rapid and reliable method to analyses the mixture on site and in real time. The aim of this research is to use a previously developed ultrasonic method to analyse mixtures of diesel and biodiesel. The samples were blends which proportion varied from 0% (pure diesel) to 20%. The ultrasonic quantities were speed of sound and ultrasonic attenuation. Those parameters were determined in a transmission/reception scheme. The measurement uncertainties were evaluated according to the Guide to the Expression of Uncertainty in Measurement (BIPM JCGM 100:2008). Complementary, commercial samples (B2 and B10) were obtained from a local supplier and evaluated accordingly. The expanded definitional uncertainty was 2.0 ms(-1) for speed of sound and 0.032 dB cm(-1) (p = 0.95). The method has shown capability to identify less than 4% of biodiesel in diesel blends for both parameters.