Biotinylated Portulaca oleracea polysaccharide (Bio-POP) conjugates were successfully prepared by the esterification reaction. The biotinylated polysaccharide products were an off-white powder with an average degree of substitution of 42.5%. After grafting biotin onto POP, the thermal stability of Bio-POP conjugates was much higher than that of POP and the surface topography of Bio-POP was a loose and porous cross-linked structure. The cytotoxicity assay in vitro demonstrated that POP, biotin, and Bio-POP conjugates exhibited different cytotoxicity to HeLa, MCF-7, LO-2, and A549, in particular POP inhibited the growth of the A549 cell line more than other cell lines. The nuclear staining method demonstrated that Bio-POP conjugates can interfere with the apoptosis of A549 cells to some extent and the immunofluorescence staining photograph illustrated that Bio-POP conjugates induced A549 cells to exhibit immune activity. Therefore, the combination of biotin and Portulaca oleracea polysaccharides had immune synergistic therapeutic effects on A549 cells and can be applied in the field of anti-tumor conjugate drugs.