In recent years, biodiesel has been demonstrated to offer a suitable level of reliability and attracted the attention of many researchers. Accordingly, various studies have been carried out to account for the biodiesel production and application, producing valuable reports and findings. In this research study, the effects of biodiesel on engine noise were studied on the basis of a time–frequency analysis. To do so, the acquired acoustic signal was initially filtered and denoised. Then the signal was transferred to the time–frequency SPL domain using short-time Fourier transform. In the A weighted signal, the SPL of all treatments were compared using an innovative visual technique. In this novel approach, the values of area percentages of the obtained SPL in the time–frequency domain were used to compare the propagated noise due to variables. The method revealed a consistent trend for all fuel blends at all engine rotational speeds. The analysis results showed that B10 (10% methyl/ethyl ester and 90% diesel fuel) and B30 had the lowest and highest A-Weighted SPL, respectively. Additionally, it was found that the engine had a maximum sensitivity for all fuel blends at an engine rotation speed of 1600 RPM. Moreover, Z-weighted (linear) signal processing was used to investigate what happens in a complete thermodynamic cycle at 1600 RPM. The developed time–frequency methodology successfully exposed all of the important acoustic events of the engine. The results of this study showed that the most effective acoustic events in engine noise were combustion, piston slap, and outlet valve closing. Furthermore, higher percentages of biodiesel blends resulted in longer combustion duration.