Introduction: Traditional Chinese medicine (TCM) constitutional theory guides clinical diagnosis and treatment. This study aimed to further explore more objective biological expressions among people with different constitutions. Methods: Antibody microarrays were used to investigate the differentially expressed proteins (DEPs) in sera of people with three different constitutions, Balanced constitution (BC) group (n = 18), Phlegm-dampness constitution (PdC) group (n = 13) and Damp-heat constitution (DhC) group (n = 13). Results: The clusters of DEPs were basically consistent with the clinical classification which is according to Classification and Determination Standards of Constitutions in Traditional Chinese Medicine. Biological functional analysis of DEPs revealed that the signaling pathways involved in PdC and DhC were JAK-STAT, TGF13/BMP and PI3K-Akt pathways. These biological pathways played their roles were basically related to cardiovascular diseases, abnormality of lipid metabolism and tumor diseases. The phenomenon of more up-regulated proteins in PdC while more down-regulated proteins in DhC suggesting that these two constitutions may be related to hyperthyroidism or low in immune system respectively. Conclusion: Our study showed that both PdC and DhC had unique serum protein profiles compared to BC. Signaling pathways related to DEPs of PdC and DhC providing evidence for the susceptibility of cardiovascular diseases and other diseases in PdC and DhC population. Our study could provide insights for future in-depth investigation of the material basis and mechanism of TCM constitutional theory.