We report the rapid single-step flame synthesis of hydrophobic carbon layers (C-layers) on the surface of stainless-steel (SS) substrates using vaporized biodiesel as the fuel. A co-flow canola methyl ester/air diffusion flame is used to generate a hydrophobic monolayer on the surface of the metal substrate upon its insertion into the reaction zone. Carbon deposition on the surface of the SS substrates varies by changing the SS disk’s position in the post-flame, and by varying its exposure time. The thickness and mass of the flame-formed monolayer varied depending on the substrate’s insertion point into the flame. However, the variation of mass did not significantly impact the C-layer’s uniformity or hydrophobicity. We hypothesize that a small “inner-cone” of the biodiesel flame along with a high soot propensity can result in an ideal medium to form uniform hydrophobic C-layers of unique hierarchical surface structure. This is supported by introducing SS substrates in methane/air flames formed using the same co-flow burner. The hydrophobic property of the carbon deposits was quantified by measuring the contact angle of water droplets placed on the film’s surface. A water droplet drop test was conducted on the flame-formed hydrophobic layers to study their wettability property.