Phenylboronic acid-functionalized chondroitin sulfate A (CSA)-deoxycholic-acid (DOCA)-based nanoparticles (NPs) are prepared for tumor targeting and penetration. (3-Aminomethylphenyl)boronic acid (AMPB) is conjugated to CSA-DOCA conjugate via amide bond formation, and its successful synthesis is confirmed using proton nuclear magnetic resonance spectroscopy (H-1-NMR). Doxorubicin (DOX)-loaded CSA-DOCA-AMPB NPs with a mean diameter of similar to 200 nm, a narrow size distribution, negative zeta potential, and spherical morphology are prepared. DOX release from NPs is enhanced at acidic pH compared to physiological pH. CSA-DOCA-AMPB NPs exhibit improved cellular uptake in A549 (human lung adenocarcinoma) cells and penetration into A549 multicellular spheroids compared to CSA-DOCA NPs as evidenced by confocal laser scanning microscopy and flow cytometry. In vivo tumor targeting and penetrating by CSA-DOCA-AMPB NPs, based on both CSA-CD44 receptor and boronic acid-sialic acid interactions, is revealed using near-infrared fluorescence (NIRF) imaging. Penetration of NPs to the core of the tumor mass is observed in an A549 tumor xenografted mouse model and verified by three-dimensional NIRF imaging. Multiple intravenous injections of DOX-loaded CSA-DOCA-AMPB NPs efficiently inhibit the growth of A549 tumor in the xenografted mouse model and increase apoptosis. These boronic acid-rich NPs are promising candidates for cancer therapy and imaging.