Mixed-mode chromatography (MMC) is a promising technology for protein separation with high adsorption capacity, good selectivity, salt-tolerance, and facile elution. Expanded bed adsorption (EBA) can capture target proteins directly from bioparticle containing crude feedstock without solid-liquid separation. In this study, MMC and ERA were combined to develop a mixed-mode EBA technique for human serum albumin (HSA) separation. Two mixed-mode EBA resins (using quartz-densified agarose beads and tungsten carbide/agarose composite beads, respectively) with. tryptamine as the ligand were prepared (TA-S and TA-T). Static and dynamic adsorption behaviors of HSA were determined under varying conditions and typical adsorption properties of pH dependence and salt-tolerance were found. The performance of the resins in expanded bed was studied, and the dynamic binding capacities at 10% breakthrough (Q(10%) ) of TA-S and TA-T at bed expansion of 1.8 were 27.54 and 18.04 mg/mL settled resin, respectively. Moreover, these two resins were used to separate recombinant HSA (rHSA) from Pichia pastoris culture broth. TA-S showed better separation performance and the purity of rHSA monomer reached to 83.3% with the recovery of 91.6% through a two-step elution process. Total content of rHSA monomer and degraded fragment was 99.8%. The results demonstrate that the combination of MMC and EBA would be a potential platform for protein capture with fewer pretreatments and high process efficiency.