Biosurfactants have been well recognized as an environmentally friendly alternative to chemical surfactants. However, their production remains challenging due to low productivity, short-term microbe stability and the potentially toxic by-products generated in the growth media. To overcome these challenges, the emerging biofilm-based biosynthesis was investigated in this study. A fresh insight into the biosynthesis process was provided through using waste fly ash as a carrier material. The biofilm produced by biosurfactant producer B. subtilis N3-1P attached onto the surface of fly ash acted as a robust and effective biocatalyst. Zeta potential analysis and scanning electron microscope (SEM) characterization were conducted to help unravel the biocatalyst formation. High-value biosurfactant products were then produced in an efficient and sustainable manner. Stimulation by a fly ash assisted biocatalyst on biosurfactant production was confirmed. The biosurfactant yield was boosted over ten times after 24 hours, at a fly ash dosage of 0.5%. The highest biosurfactant yield was achieved after five days, with a final productivity of 305 critical micelle dilution. The underlying mechanism of fly ash assisted biosurfactant production was tracked through it exerting an effect on the quorum sensing system. Fourier-transform infrared (FTIR) spectroscopy and matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) analysis demonstrated that the final biosurfactant product belonged to the lipopeptides. This research output is expected to accelerate the development of more effective bioreactors, and make a continuous contribution to high-value product generation and waste reduction.