β-glucan, a polysaccharide found in various sources, exhibits unique physicochemical properties, yet its high polymerization limits clinical applications because of its solubility. Addressing this limitation, we introduce PPTEE-glycan, a highly purified solubleβ-1,3/1,6-glucan derived from Aureobasidium pullulans. The refined PPTEE-glycan demonstrated robust immune stimulation in vitro, activated dendritic cells, and enhanced co-stimulatory markers, cytokines, and cross-presentation. Formulated as a PPTEE + microemulsion (ME), it elevated immune responses in vivo, promoting antigen-specific antibodies and CD8+ T cell proliferation.Intratumoral administration of PPTEE + ME in tumor-bearing mice induced notable tumor regression, which was linked to the activation of immunosuppressive cells. This study highlights the potential of high-purity Aureobasidium pullulans-derived β-glucan, particularly PPTEE, as promising immune adjuvants, offering novel avenues for advancing cancer immunotherapy.