Sono-enzymatic biodiesel synthesis from different feedstocks has been significantly investigated in recent years, which reports the impact of ultrasound (US) on the structure of lipases. However, the links between alteration in the structure of lipases due to US and its activity enhancement have yet to be established. This study reveals the molecular mechanism of US-induced enhancement of lipase-catalyzed reactions in biodiesel synthesis. Using molecular simulations, we first analyzed the 3D structures and amino acid (aa) composition of the binding pockets of two lipases, viz. CALB and TLL. Next, docking analysis of different ligands (substrates and products of the reactions) with lipases was performed to reveal the interacting residues. We identified the US-induced alteration in the secondary structure of lipase from previous reports. Our analysis revealed that most binding pockets are located in the α-helix and random coil motifs, and binding interactions are mainly hydrogen bonding and hydrophobic interactions. We observed a rise in α-helix and random coils of lipase with sonication. The net effect of sonication is the widening of the catalytic cavity, thus easy substrate accessibility to the active site. Thus, enhanced enzymatic activity is manifested as faster kinetics of the reactions with higher yields.