The rheological behavior of a canola oil-based bio-lubricant was correlated with its surface activities using a rotary rheometer. The experiments on the gap size, substrate, and surface conditions led to a consistent and conceivable assumption that the self-assembled monolayer was extended to a degree that was sufficient to make a significant change in the bulk properties. In parallel, the thermal behavior was also connected to the surface activities. Differential scanning calorimetry was used to measure what is thought to be the monolayer melting point without using a graphite substrate. Crystallization points were measured at relatively high temperatures. Tribological tests were conducted and explained based on the rheological and thermal findings. A scenario describing both the boundary and bulk regimes was hypothesized. Rheometry and thermal analysis techniques are promising because of the variety of variables that could be controlled, such as temperature, shear, time, gap and substrate. To the best of our knowledge, this is the first study using macroscopic rheology to study the surfactant activities of vegetable oil. The influence of substrate and gap size on rheological behavior could change the current standards for using rotary rheometers.