In this paper, an experimental investigation has been carried out on the acoustic characteristics of a compression ignition (CI) engine running with biodiesel blends under steady state operating conditions. The experiment was conducted on a four-cylinder, four-stroke, direct injection and turbocharged diesel engine which runs with biodiesel (B50 and B100) and pure diesel. The signals of acoustic, vibration and in-cylinder pressure were measured during the experiment. To correlate the combustion process and the acoustic characteristics, both phenomena have been investigated. The acoustic analysis resulted in the sound level being increased with increasing of engine loads and speeds as well as the sound characteristics being closely correlated to the combustion process. However, acoustic signals are highly sensitive to the ambient conditions and intrusive background noise. Therefore, the spectral subtraction was employed to minimize the effects of background noise in order to enhance the signal to noise ratio. In addition, the acoustic characteristics of CI engine running with different fuels (biodiesel blends and diesel) was analysed for comparison. The results show that the sound energy level of acoustic signals is slightly higher when the engine fuelled by biodiesel and its blends than that of fuelled by normal diesel. Hence, the acoustic characteristics of the CI engine will have useful information for engine condition monitoring and fuel content estimation.