The objective is to use orthogonal experiment to optimize the pretreatment on the determination of serum cholesterol and its markers by GC-MS. And then the method is evaluated in a methodological perspective. The methodis to Use L16(211) orthogonal experiment design to observe the influence of three key steps, althogether seven factors of pretreatment, which are saponification (KOH ethanol solution concentration, temperature and time), extraction (dose) and derivatization (temperature , time and dose). As for the results, the conditions of optimal pretreatment are as follows： the ethanol solution is 1 mol·L-1 KOH, the saponification temperature is 70 ℃；the saponification time is 60 min；the Solvent quantity is 2 mL； the derivatization temperature is 70 ℃；the derivatization time is 60 min, and the derivatization agent is 100 μL. Through the optimization by orthogonal design and methodological evaluation, the determination of serum cholesterol and its markers by GC-MS is excellent in terms of accuracy and precision, and methodological evaluation indexes are better than those reported in other papers.