Surfactants are one of the versatile products of the chemical industry owing to their inherent nature to lower the surface and interfacial tension and interface layer stabilization but restricted due to non-degradability issues and hazardous effects. For combating such problems, bio-based surfactants (Biosurfactants) could be potential alternative. The biosurfactants are an integral part of life as they have been explored in pharmaceutical sectors, therapeutics, health, food products, health and beauty products, and agriculture, creating sustainable economics and promoting sustainable development goals. Therefore, researchers have been interested in producing, optimizing, and utilizing biosurfactants for various applications. In the present work, biosurfactants were prepared from three different feeds, namely Jatropha carcus, Sapium sebiferum, and used cooking oil (UCO) using sodium bisulfite (NaHSO 3) as sulfonating reagent and alumina (Al 2 O 3 )as the heterogeneous catalyst to yield 70 %, and comparative assessment was done. The biosurfactant production involved a two-step process viz. biodiesel production and biosurfactant production thereof. The biodiesel produced in the first step met all the BIS 15607 : 2016 specifications and both products were confirmed through analytical techniques like Fourier transform infrared (FTIR) and Nuclear magnetic resonance (NMR) spectroscopy. Further, the produced biosurfactants were employed to reduce the biodiesel-glycerol layer separation time, and a significant time reduction in the biodiesel-glycerol separation was found.