Reliability on the observed results of biodiesel prepared from high viscous rubber seed oil (RSO) using fluorite and calcined limestone as catalysts has been studied using uncertainty error analysis. In this study, the error analysis was performed for three different deviations 5, 10, and 15% of optimum process parameter conditions of 12:1 methanol: oil molar ratio (mol/mol), 4 (wt %) of catalyst concentration, and 5(h) of reaction time, respectively. All the uncertainty experiments were performed for upper level and lower level of every deviation mentioned with. From these experiments, it was observed that methanol: oil molar ratio (mol/mol) shows more error on a small deviation of optimum conditions when compared with catalyst concentration and reaction time, respectively. It is also observed that molar ratio is the most sensitive process parameter in biodiesel preparation from RSO using fluorite and calcined limestone as heterogeneous base catalysts when compared to catalyst concentration and reaction time, respectively.