A nanoplatform carrying doxorubicin (Dox) for cancer therapy and a dye for imaging was developed based on a natural triple helix beta-glucan (t-LNT) and polydeoxyadenylic acid (poly(dA)). The t-LNT-Dox conjugates were prepared through Schiff-base reaction between the aldehyde group in the oxidized t-LNT and the amino group of Dox, the single chains (s-LNT-Dox) of which interacted with the poly(dA)-dye to form a composite s-LNT-Dox/poly(dA)-dye through hydrogen bonding between s-LNT and poly(dA). t-LNT-Dox was confirmed to acid-responsively release Dox in vitro, showing enhanced cytotoxicity against HeLa cancer cells with time. It was confirmed that Dox and the dye could be simultaneously delivered into HeLa cells or the tumors with a prolonged duration time. Furthermore, LNT-Dox conjugates effectively inhibited tumor growth and decreased adverse effects of the free Dox in vivo. Hence, this work develops a new strategy to fabricate the nanoplatform for therapy and imaging using a natural polysaccharide.