The use of lignocellulosic biomass to create natural flavor has drawn attention from researchers. A key flavoring ingredient that is frequently utilized in the food industry is vanillin. In this present study, Pediococcus acidilactici PA VIT effectively involved in the production of bio-vanillin by using Ferulic acid as an intermediate with a yield of 11.43 µg/mL. The bio-vanillin produced by Pediococcus acidilactici PA VIT was examined using FTIR, XRD, HPLC, and SEM techniques. These characterizations exhibited a unique fingerprinting signature like that of standard vanillin. Additionally, the one variable at a time method, placket Burmann method, and response surface approach, were employed to optimize bio-vanillin. Based on the central composite rotary design, the most important process factors were determined such as agitation speed, substrate concentration, and inoculum size. After optimization, bio-vanillin was found to have tenfold increase, with a maximum yield of 376.4 µg/mL obtained using the response surface approach. The kinetic study was performed to analyze rate of reaction and effect of metal ions in the production of bio-vanillin showing Km of 10.25, and Vmax of 1250 were required for the reaction. The metal ions that enhance the yield of bio-vanillin are Ca 2+ , k + , and Mg 2+ and the metal ions that affects the yield of bio-vanillin are Pb + and Cr + were identified from the effect of metal ions in the bio-vanillin production.