Chronic heat stress (CHS) reduces the production efficiency of the buffalo dairy industry. Relatively low-abundance proteins with particular functions in biological processes are changed by CHS. The present study aimed to quantify the differences in low-abundance proteins of crossbred dairy buffaloes under CHS and thermal-neutral (TN) conditions. With label-free quantification, 344 low-abundance proteins were identified in serum. Of these, 17 differentially expressed low-abundance proteins with known functions were detected, and six of the differentially expressed proteins related to heat stress were validated with parallel reaction monitoring. Lipase (LPL), glutathione peroxidase 3 (GPX3), cathelicidin-2 (CATHL2), ceruloplasmin (CP), and hemoglobin subunit alpha 1 (HBA1) cooperatively played roles in the thermal fitness of dairy buffalo by decreasing heat production and increasing blood oxygen delivery. Also, dairy buffaloes may adapt to CHS and hypoxia with high levels of RBCs, HBA1 and CP to increase blood oxygen delivery capacity.