This study was designed to investigate the changes in acoustic properties of whole blood during the coagulation process. High frequency (from 20 to 40 MHz) ultrasound parameters were measured both in double transmission (DT) and backscattering (BS) mode to assess sound velocity and backscatter coefficient, respectively. The integrated backscatter coefficient (IBC) and the effective scatterer size (ESS) were deducted from the backscatter coefficient. Measurements were performed on whole blood samples collected from 12 healthy volunteers. During the blood clotting process (2 h observation), acoustic parameters were measured with 15 s time resolution for the transmission parameter and 5 s (for the 5 first min) and 30 s (for the end of the observation time) for the backscattering parameters. The results obtained clearly showed that simultaneous measurements of parameters in DT and BS modes are able to identify several stages during the in vitro blood clotting process. In particular, red blood cell (RBC) aggregation can be described from the backscattering parameters and liquid-gel transition phase of blood from the sound velocity. Intra- and inter-individual dispersion of these parameters were also measured and discussed.