Abstract: The accurate and quantitative analysis of low-abundance proteins in complex matrices has always been the focus and faced difficulty in protein research. With the rapid development of mass spectrometry technology, isotope dilution mass spectrometry has become an important method for accurate quantitative analysis of low-abundance proteins in serum. In this study, isotopically labeled human growth hormone was used as an internal standard. A strategy for separation of serum samples was established based on off-line two-dimensional high-performance liquid chromatography (2D HPLC). Then, combined with high performance liquid chromatography-isotope dilution mass spectrometry (IDMS) method, quantitative analysis of lowabundance protein growth hormone (hGH) in human serum was performed with high accuracy. Based on the measurement and uncertainty calculation of the simulated samples (blank serum with known quality of growth hormone standard substance, theoretical concentration 12.00 ng/g) and international comparison samples, the quantitative results are (11.45±2.33) ng/g and (12.84±1.46) ng/g, respectively. This method has high accuracy and good repeatability, and can provide an effective measurement method for accurate quantitative analysis of low-abundance proteins in complex matrices. The results can trace to reference material.