AbstractUsing readily available biomass, it is possible to produce fatty acid methyl esters (FAME) at a low cost. This study focused on the transesterification process of waste frying vegetable oil to generate FAME (biodiesel) using a cost‐effective heterogeneous nano‐catalyst synthesized from waste cow teeth (CT). The cow teeth nano‐catalyst (CTNC) was synthesized via a three‐step method of calcination, hydration‐dehydration, and re‐calcination. The synthesized CTNC samples were characterized. The Box–Behnken design of response surface methodology (RSM) was used to obtain the optimal operating conditions that maximize the FAME yield (Y 1 ) and the effects of the transesterification process conditions on FAME production. The results revealed that CTNC is of microporous structure with higher crystallinity. It consists of hydroxyapatite, beta‐tricalcium phosphate, and calcium oxide, having a mean particle size of 43.96 nm, a specific surface area of 124.77 m 2 /g, and a pore volume of 0.303 cm 3 /g. The process conditions that significantly influenced the EWFVOME yield were the CTNC loading, reaction time, and MTOR. A maximum of 98.70% (Y 1 ) was obtained at the optimum transesterification process variables of reaction time (2 h), MTOR (12:1), and CTNC loading (3.75 wt%). The biodiesel fuel properties were found to be within the acceptable values of ASTM D6751 and EN 14214 fuel standards.