Human milk provides a range of nutrients and bioactive components, which can support the growth and development of infants. However, human milk composition may change due to geographic and ethnic variation. This study investigated the variation of the Chinese human milk serum proteome based on mothers with different ethnicities living in different parts of China, using TMT labeling combined with Nano-LC Q Exactive HF MS/MS proteomics. In total, 693 proteins were identified and quantified in human milk serum from Yunnan (Han and Bai ethnicity), Gansu (Han and Tibetan ethnicity), Xinjiang (Uygur ethnicity), and Inner Mongolia (Mongolian ethnicity). The biological function distribution of identified proteins and the summed intensity of proteins belonging to each biological function were similar among groups. The five relatively highly abundant milk serum proteins, lactoferrin, serum albumin, polymeric immunoglobulin receptor, macrophage mannose receptor 1, and bile salt-activated lipase were not significantly different among different geographies and ethnicities. On the other hand, we found 34 proteins that did significantly differ with geography and ethnicity. Those significantly different proteins have known strong interaction in inflammation response and regulation of multi-organism processes. Taken together, biological function distribution was similar on both the qualitative and quantitative levels, and proteins with similar abundance are important in providing basic nutrition and protection for infants, whereas the significantly different proteins may be important for the healthy development of infants from different locations and ethnicities.