The uncertainty of GC method in the determination of fatty acids in peanut oil was evaluated based on Monte Carlo method (MCM). The results show thatthe uncertainty of C16:0 is 10.78%±0.23%, C18:0 is 3.52%±0.08%, C18:1n9c is 45.75%±0.58%, C18:2n6c is 32.10%±0.52%, C22:0 is2.84±0.07%. The MCM method avoids the complicated measurement deduction such as partial derivative of measurement model and the process of type A and type B evaluation when used to evaluate the measurement uncertainty. The uncertainty of measurement of 21 fatty acid components in peanut oil can be easily and quickly calculated by MCM software.This method provides technical support for improving the evaluation ability of laboratory uncertainty.